Amino acid derivatives, methods of preparing said derivatives and antihypertensive drugs containing them

ABSTRACT

The present invention relates to an amino acid derivative represented by the formula ##STR1## wherein R 1  and R 2  represent hydroxyl, alkyloxy, aryloxy, arylkyloxy, alkyl, aryl, or aralkyl; R 3 , R 5 , R 8  and R 11  are hydrogen or alkyl; R 4 , R 7  and R 10  are hydrogen, or substituted or unsubstituted alkyl, aryl or aralkyl; R 6  and R 9  are hydrogen, alkyl, aryl, or aralkyl; R 12  is hydroxyl, alkyloxy, aryloxy, aralkyloxy, amino, mono- or di-alkyl-, aryl- or aralkyl amino; and R 6  and R 7  combined together, and R 9  and R 10  combined together independently may form a substituted or unsubstituted alkylene bridge. The present invention also relates to a method of preparing said amino acid derivatives, and to anti-hypertensive drugs containing them.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel amino acid derivatives andantihypertensive drugs containing them. The present invention alsorelates to methods of preparing these derivatives.

2. Detailed Description of the Invention

The present inventors have succeeded in the synthesis of novel aminoacid derivatives represented by the general formula: ##STR2## Moreover,the present inventors have found that these derivatives, having anantihypertensive activity, are useful as antihypertensive drugs andintermediates for their production, and, based on this finding, havecompleted the present invention.

R₁ and R₂ in the foregoing formula, being the same or different fromeach other, each individually represents hydroxyl, alkyloxy, aryloxy,aralkyloxy, alkyl, aryl, or aralkyl.

The alkyloxy is a lower alkyloxy having 1˜5 carbon atoms such asmethoxy, ethoxy, n-propyloxy, n-butyloxy, and n-pentyloxy.

The aryloxy is the one having 6˜12 carbon atoms such as phenoxy,naphthyloxy, and p-tolyloxy.

The aralkyloxy is the one having 6˜12 carbon atoms such as benzyloxy,phenetyloxy, phenylpropyloxy, and hydroxybenzyloxy.

The alkyl is a lower alkyl having 1˜5 carbon atoms such as methyl,ethyl, n-propyl, n-butyl and n-pentyl.

The aryl is the one having 6˜12 carbon atoms such as phenyl and p-tolyl.

The aralkyl is the one having 6˜12 carbon atoms such as benzyl,phenethyl, and phenylpropyl.

R₃, R₅, R₈, and R₁₁ at least two of which are the same or all of whichare different from one another, each individually represents hydrogenatom, or alkyl: for example, the one having 1˜5 carbon atoms such asmethyl, ethyl, n-propyl, n-butyl, and n-pentyl. That is to say, all ofR₃, R₅, R₈, and R₁₁ may each individually represent hydrogen atom ormethyl, or R₃ and R₅ may each individually represent hydrogen atom, andR₈ and R₁₁ may individually represents methyl; all of them may bedifferent from one another: for example, R₃ represents hydrogen atom, R₅represents methyl, R₈ represents ethyl, and R₁₁ represents isopropyl.

R₄, R₇, and R₁₀ at least two of which are the same or all of which aredifferent from one another, each individually represents hydrogen atom,or substituted or unsubstituted alkyl, aryl, or aralkyl.

The alkyl represents a lower alkyl having 1˜5 carbon atoms such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, andn-pentyl.

The aryl represents the one having 6˜12 carbon atoms such as phenyl,p-tolyl, and naphthyl.

The aralkyl represents the one having 6˜12 carbon atoms such as benzyland naphthylmethyl.

Substituent groups in the foregoing substituted groups may include, forexample, hydroxyl, carboxyl, carbamoyl, amino guanidino, imidazolyl,indolyl mercapto, and lower alkylthio.

R₆ and R₉, being the same or different from each other, eachindividually represents hydrogen, alkyl, aryl, or aralkyl. The alkyl,the aryl, and the aralkyl, being, for example, the foregoing ones, mayfurther include alicylic compounds such as cyclopropyl, cyclopentyl, andcyclohexyl; and aromatic ring-condensed alicyclic compounds such as2-indanyl and 1-indanyl.

R₁₂ represents hydroxyl, alkyloxy, aryloxy, aralkyloxy, amino, mono- ordi-alkyl-, aryl-, or aralkylamino. The alkyloxy, the aryloxy, thearalkyloxy, the alkyl, the aryl, and the aralkyl may each individuallyinclude, for example, those cited above as examples of R₁ and R₂.

R₆ and R₇, combined together, may form an alkylene bridge having 2˜4carbon atoms, an alkylene bridge having 2˜3 carbon atoms and 1 sulfuratom, an alkylene bridge having 3˜4 carbon atoms which contains a doublebond, or substituted ones of these bridges; R₉ and R₁₀, combinedtogether, may form the same alkylene bridges.

Examples of substituents on the foregoing bridges include hydroxyl,lower alkoxy, lower alkyl, oxo(o═), amino, condensed allyl, a condensedaromatic ring, and a condensed alicyclic ring.

Structures formed by such alkylene bridges may be, for example, thefollowing ones: ##STR3##

Amino acids constituting the amino acid derivatives of the presentinvention may be either the L-isomer or the D-isomer.

The amino acid derivatives of the present invention may be in the formof a salt, such as a metal salt, as for example, sodium, potassium,lithium, and calcium salts or a salt with an organic base. As theorganic base, there can be adopted amines such as ammonia (ammoniumsalt), dicyclohexylamine, and N-methyl-D-glucamine; and basic aminoacids such as lysine and arginine.

When the amino acid derivatives are to be included in theantihypertensive drugs of the present invention, they need to be in aform of a pharmaceutically acceptable salt.

The amino acid derivatives of the present invention are tripeptidederivatives of which the terminal amino group is combined with aphosphorus compound (P), being represented by the formula: P-X-Y-Z (X,Y, and Z each represents an amino acid residue).

The amino acid corresponding to X typically includes alanine, leucine,isoleucine, glycine, phenylalanine, arginine, glutamic acid, glutamin,lysine, valine, ornithine, methionine, serine, and threonine; the aminoacid corresponding to Y typically includes proline, hydroproline,glycine, N-substituted glycine, and thioproline; the amino acidcorresponding to Z typically includes proline, hydroxyproline, glycine,alanine, serine, aspartic acid, arginine, tyrosine, phenylalanine,valine, leucine, isoleucine, threonine, methionine, glutamic acid,glutamine, lysine, cystine, tryptophan, and histidine.

When the tripeptide derivative has functional groups, the functionalgroups may be protected by protecting groups ordinarily used insynthetic chemistry of peptides; the protected derivatives are alsoencompassed by the amino acid derivatives of the present invention.

Examples of methods for preparation are as follows:

Method 1: ##STR4## wherein R₁˜12 each individually represents the sameas defined above.

The desired compound can be prepared by causing a phosphoridate [III]such as phosphochloridatediester and phosphochloridatemonoester to reactwith, for example, a hydrochloride derivative of ester or amide of thetripeptide derivative [II] in an inert solvent such as methylenechloride in the presence of a base such as triethylamine to transformthe N-terminal of an the tripeptide derivative into a phosphorusderivative, followed, if necessary, by selectively removing protectinggroups by means of hydrolysis with an alkali, catalytic hydrogenation,or the like.

On the other hand, the desired compound can also be prepared by directlycondensing a phosphoridate [III] with a free compound of the tripeptidederivative [II].

Method 2: ##STR5## wherein R₁˜12 each individually represents the sameas defined above.

The desired compound can be prepared by subjecting a phosphorusderivative of a dipeptide [IV] and an amino acid derivative [V] such asa hydrochloride of ester or amide to a condensation reaction in an inertsolvent such as dimethylformamide (DMF) with the use of a condensingagent such as N,N'-dimethylaminopropylethylcarbodiimide (WSC) and1-hydroxybenzotriazole (HOBt) to obtain a phosphorus derivative of thetripeptide [I], followed, if necessary, by selectively removingprotecting groups by means of hydrolysis with an alkali or catalytichydrogenation.

Method 3: ##STR6## wherein R₁˜12 each individually represents the sameas defined above.

The desired compound can be obtained by subjecting a phosphorusderivative of an amino acid [VI] and a dipeptide derivative [VII] suchas a hydrochloride of ester or amide to a condensation reaction in aninert solvent such as dimethylformamide with the use of a peptidecondensating agent used in synthetic chemistry of peptides, such as WSCand HOBt, to obtain a phosphorus derivative of a tripeptide, followed,if necessary, by selectively removing protecting groups by means ofhydrolysis with an alkali or catalytic hydrogenation.

The methods will be illustrated with more specific examples below.

A substituted phosphoryl derivative can be prepared by preparing apeptide derivative represented by the general formula: ##STR7## whereinZ represents the same as defined above, followed by causing the peptidederivative to react with a substituted phosphoryl halogenide such asdiethyl- or dibenzylphosphoryl chloride. The substituted phosphorylderivative, for example, dibenzylphosphoryl chloride, can be convertedinto a phosphoryl derivative by subjecting it to hydrogenation usingpalladium carbon as catalyst.

The foregoing tripeptide, an intermediate for the derivatives of thepresent invention, can be prepared by causing an alanine having aprotected amino group to react with a proline having a protectedcarboxyl group to prepare alanylproline, removing the protecting groupsof the alanylproline, and causing the resulting product to react withthe Z component, an amino acid having a protected amino group, followed,if necessary, by removing the protecting group.

On the other hand, the tripeptide can also be prepared by preparingdibenzylphosphorylalanylproline, follwed by causing the product to reactwith the foregoing selected amino acid ester according to the method ofJapanese patent first publication No. 104863/1981.

An alanylproline derivative of which the constituent proline residue hasa hydroxyl group (hydroxyproline) can also be prepared in the samemanner.

Those protecting groups for amino, imino, carboxyl, and hydroxyl groups,those methods for the protection, those methods for removal of theprotecting groups, and those methods for amide-linking by condensationbetween amino and carboxyl groups which are all employed in thepreparation of the derivatives of the present invention andintermediates therefore may be the ones ordinarily used in methods forpeptide synthesis or generally or conventionally employed in the knownliterature, for example, Protein Chemistry, 1 Amino Acid.Peptide, e.g.pages 405˜509 (1969), compiled by Shiro Akabori, Takeo Kaneko, and KozoNarita, published by Kyoritsu Shuppan Co. An amide-linkage isadvantageously formed from an amino acid by a condensation method inwhich an active ester of the amino acid having a protected amino group,such as p-nitrophenylester and N-hydroxysuccinimideester, is used forreaction. When a solvent is used in the reaction, DMF or water can beadopted as solvent. The reaction temperature may be a room temperatureor so, but the reaction can also, as needed, be accelerated by heating.

The derivatives of the present invention are isolated from the reactionmixture by concentrating the reaction mixture to dryness, purifying theresidue by means of column chromatography, followed by lyophilization ofthe product.

When the derivatives of the present invention are used, as an activeingredient, for an antihypertensive drug, there may be adopted theirfree forms, their nontoxic forms of salt, or their nontoxic forms havingprotecting groups. An amino acid constituting the derivatives for use asan antihypertensive drug of the present invention may be either theL-isomer or the D-isomer.

The amino acid derivatives of the present invention are useful as anantihypertensive drug for treating hypertensive mammals includinghumans. The derivatives can be used for lowering blood pressure byformulating them into a preparation such as tablets, capsules, andelixirs for oral administration and into an aspetic liquid preparationor an aseptic suspension preparation for parenteral administration. Theamino acid derivatives of the present invention can be administered to asubject necessitating such treatment (animals and humans) in a dosagerange of 0.2˜500 mg per subject generally several times a day, that is,in a total daily dosage of 1˜2000 mg. The dosage varies according to theseriousness of disease, the body weight of subjects, and other factorsacknowledged by those skilled in the art.

The amino acid derivatives of the present invention can also beadministered together with diuretics or other antihypertensive drugs.Typically, these drugs are administered in a dosage combination of whichone unit of daily dose is in the range from 7/8 times as large as aclinical dosage minimally recommended, to a level maximally recommendedsingly for each entity of disease. These combinations are speciallyshown as follows: A kind of antihypertensive drugs of the presentinvention which is clinically effective in a daily dosage range of15˜200 mg can effectively be administered together with the followingother antihypertensive drugs and diuretics in a daily dosage range of3˜200 mg: hydrochlorothiazide (15˜200 mg), chlorothiazide (125˜2000 mg),ethacrynic acid (15˜200 mg), amiloride (5˜20 mg), furosemide (5˜80 mg),propranolol (20˜480 mg), timolol (5˜50 mg), methyldopa (65˜2000 mg). Theforegoing dosage ranges are adjusted on the basis of unit according tothe necessity for the possible daily divided dosage. The dosage variesaccording to the seriousness of disease, the body weight of subject, andother factors acknowledged by those skilled in the art.

The foregoing typical combinations of drugs are formulated intopharmaceutical compositions stated below. About 0.2˜500 mg of thederivatives of the present invention, pharmaceutically acceptable saltcompounds, or mixtures of both are blended into unit dosage formsgenerally acknowledged or required for the pharmaceutical practicetogether with pharmaceutically acceptable vehicles, carriers,excipients, binders, antiseptics, stabilizers, flavorings, and so forth.The amount of each active substance in these compositions orpreparations is adjusted in such a way as to give an appropriate dosageof the prescribed range.

Specific materials which can be incorporated into tablets, capsules, andso forth are as follows: A binder such as traganth, gum arabic,cornstarch, and gelatin; an excipient such as microcrystallinecellulose; a swelling agent such as cornstarch, pregelatinized starch,and arginic acid; a lubricant such as magnesium stearate; a sweetnersuch as sucrose, lactose, and saccharin; a flavoring such as peppermint,an oil from Gaultheria adenothrix Maxim, and cherry. When the unitdosage form of the preparation is a capsule, a liquid carrier such asfatty oil can further be incorporated in the foregoing type materials.Various other materials can be present as a coating material or in orderto vary the physical form of unit dosage forms according to othermethods. For example, tablets can be coated with shellac and/or sugar.Syrups or elixirs can contain active compounds, sucrose as a sweetner,methyl- and propylparaben as an antiseptic, a coloring matter, aflavoring such as cherry and an orange flavoring.

Aseptic compositions for injection can be formulated according to theusual practice for preparation of pharmaceutical dosage forms, in whichpractice an active substance is dissolved or suspended in a vehicle suchas water for injection; natural vegetable oils such as sesame oil, palmoil, peanut oil, and cotton seed oil; and synthetic fat vehicle such asethyl oleate. A buffer, an antiseptic, and an antioxidant can further beincorporated as occasion demands.

The present invention will be explained precisely in the followingExamples.

Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth therein.

EXAMPLES Example 1

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-proline L-arginine salt:

A. Dibenzylphosphorylchloride

A phosphorus trichloride (3.7 g, 25 m mole) in benzene (15 ml) solutionwas added dropwise and slowly, to the mixture of dimethylaniline (6.1 g,50 m mole) and benzylalcohol (5.4 g, 50 m mole) at a temperature of lessthan 15° C. by cooling with coolant of -15° C. and stirring. After theaddition, the mixture was further stirred for 30 minutes. To themixture, benzylalcohol (2.7 g, 25 m mole) was further added dropwise andthe mixture was reacted at a room temperature overnight.

The reaction solution was shaken with water (15 ml), and the thusobtained organic phase was separated and then washed with water, 5Naqueous ammonia and water in this order. The thus obtained mixture wasdried over anhydrous sodium sulfate. The drying agent was removed byfiltration and the remaining filtrate was distilled under reducedpressure leaving a non-coloured and oily dibenzylhalogen phosphiteproduct (3.2 g).

The thus obtained oily material gave one spot by thin layerchromatography with silica gel (developing solvent;chloroform:ethanol:ethyl acetate=5:2:5, colour forming agent:Iodine).

Dibenzyl hyddrogen phosphite (6.7 g) was dissolved in carbontetrachloride (70 ml) dried previously, and put in a 3-neck flask.Sulfurylchloride (3 g) in carbon tetrachloride (10 ml) solution wasadded dropwise to the above mixture at a temperature of less than 10° C.while cooling with coolant of -15° C. and stirring and at an atmosphereof nitrogen gas.

After the addition of sulfurylchloride, the mixture was stirred for 1.5hours at room temperature at an atmosphere of nitrogen gas. It wasconfirmed that the starting material was completely consumed by tracingthe reaction product by thin layer chromatography (developing solvent;chloroform:ethanol:ethyl acetate=5:2:5, colour forming agent:Iodine).Thus produced dibenzylphosphoryl chloride without further purificationwas used in the following reaction.

B. N-t-butyloxycarbonyl-L-alanyl-L-proline benzyl ester

N-t-butyloxycarbonyl-L-alanine (3.8 g, 20 m mole), L-proline benzylester hydrochloride (5 g, 20.6 m mole) and 1-hydroxy benzotriazole,which is hereinafter referred to as HOBt (2.7 g, 20 m mole) weresuspended in tetrahydrofuran, which is hereinafter referred to as THF(50 ml). N,N'-dimethylaminopropylethylcarbodiimide, which is hereinafterreferred to as WSC (3.8 ml) in THF (10 ml) solution was added dropwiselyto the above suspended solution while cooling a -15° C. and stirring.The reaction was carried for 3 hours at a temperature of less than 0° C.and thereafter at room temperature overnight. The thus obtained solutionwas distilled under reduced pressure leaving a residue. The residue wasdissolved in ethyl acetate and washed with 1N hydrochloric acid, water,5% aqueous sodium bicarbonate and water in order, and dried overanhydrous sodium sulfate. The thus obtained solution was distilled underreduced pressure leaving a residue. The residue was recrystallized inether-n-hexan to give N-t-butyloxycarbonyl-L-alanyl-L-proline benzylester (6.7 g, yield: 89%) having melting point of 71° to 72° C. Thisproduct gave one spot by thin layer chromatography (developing solvent;chloroform:methanol:acetic acid=95:5:3, colour forming method; sprayingwith 0.1% ninhydrin and heating).

C. N-t-butyloxycarbonyl-L-alanyl-L-proline

N-t-butyloxycarbonyl-L-alanyl-L-proline benzyl ester (6.4 g, 17 m mole)was dissolved in methanol (100 ml). Hydrogen was passed for 3 hoursthrough the solution in the presence of 10% palladium-carbon as acatalyst. The catalyst was removed by filtration and the filtrate wasdistilled under reduced pressure leaving a residue. The residue wascrystallized in ethyl acetate-n-hexan to giveN-t-butyloxycarbonyl-L-alanyl-L-proline (4.5 g, yield: 92.4%) havingmelting point of 155° to 157° C. and specific rotatory power [α]_(D) ²⁵=-90.5° (C=1, ethanol). The product gave a single spot on thin layerchromatography.

D. N-t-butyloxycarbonyl-L-alanyl-L-prolyl-L-proline benzyl ester

N-t-butyloxycarbonyl-L-alanyl-L-proline (4.3 g, 15 m mole), L-prolinebenzyl ester hydrochloride (3.7 g, 15.3 m mole) and HOBt (2.0 g, 15 mmole) were dissolved in methylene dichloride (40 ml). WSC (2.8 ml) wasadded dropwise to the above mixture while cooling to -15° C. andstirring. The reaction was carried out for 3 hours at a temperature ofnot more than 0° C., and then overnight at room temperature. The solventwas removed by distillation under reduced pressure leaving a residue.The residue was dissolved in ethyl acetate, and washed with 1Nhydrochloric acid, water, 5% sodium bicarbonate and water, in order. Themixture was dried over anhydrous sodium sulfate. The solvent thusobtained solution was distilled under reduced pressure leaving aresidue. The residue was crystallized with a mixture of ethyl acetateand n-hexan to give a crystal ofN-t-butyloxycarbonyl-L-alanyl-L-prolyl-L-proline benzyl ester (6.3 g,88.7%) having melting point of 143° to 145° C. and specific rotatorypower of [α]_(D) ²⁵ =-131.0° (C=1, chloroform).

E. Dibenzylphosphoryl-L-alanyl-L-prolyl-L-proline benzyl ester

N-t-butyloxycarbonyl-L-alanyl-L-prolyl-L-proline benzyl ester (6.0 g,12.7 m mole) was dissolved in 4.8N hydrogen chloride in dioxan (15 ml)under shaking and stirred for 40 minutes at room temperature. Thesolvent was distilled off under reduced pressure and dried ethyl esterwas added thereto. Thus obtained precipitate was obtained on the paperfilter and soon put into the desiccator containing sodium hydroxide tobe dried. All amount of the precipitate was dissolved in DMF (30 ml) andcooled to a temperature of -5° C. Triethyl amine was added to the coldmixture to neutralize it. Dibenzylphosphoryl chloride in carbontetrachloride solution synthesized in the same manner described aboveand triethyl amine were slowly and dropwise added to the aboveneutralized solution at not more than 5° C. while cooling and stirring.In such case, the PH value of the reaction solution was alwaysmaintained from 8 to 9. After the completion of the addition, thereaction was carried out at room temperature overnight. The thusobtained solution was distilled under reduced pressure leaving aresidue. To thus obtained residue ethyl acetate (300 ml) was added andthe mixture was washed with 1N hydrochloric acid, water, 5% sodiumbicarbonate, and water in order and then dried with anhydrous sodiumsulfate. The solution was distilled under reduced pressure leaving anoily dibenzylphosphoryl-L-alanyl-L-prolyl-L-proline benzyl estermaterial (7.7 g, 96%). The oily material gave a single spot with R_(f)=0.65 by the thin layer chromatography (developing solvent;chloroform:methanol:acetic acid=95:5:3, colour forming method; sprayingof 25% hydrobromic acid and 0.1% ninhydrine and heating).

F. Dibenzylphosphoryl-L-alanyl-L-prolyl-L-proline L-arginine salt

The oily material obtained above was dissolved in a mixture (20 ml) ofacetone and methanol (1:1), and 1N sodium hydroxide (15 ml) was addedthereto under cooling with ice-bath. The mixture was stirred for 1.5hours at room temperature and was neutralized with 1N hydrochloric acid.The organic solvent was distilled off under reduced pressure. Theresidue was dissolved in ethyl acetate and washed with 1N hydrochloricacid and water, and dried with anhydrous sodium sulfate. The solvent wasdistilled off under reduced pressure to give oilydibenzylphosphoryl-L-alanyl-L-prolyl-L-proline (5.7 g). The oilymaterial gave a single spot with R_(f) of 0.5 by the thin layerchromatography (developing solvent; chloroform:methanol:aceticacid=95:5:3, colour forming method; spraying of 25% hydrobromic acid and0.1% ninhydrine, and heating).

The oily material (660 mg) was dissolved in ethanol (5 ml) and anaqueous L-arginine solution (1.2 ml) was added thereto. The argininesolution has been prepared by dissolving L-arginine in the water in aratio of 1.74 g of L-argine in 10 ml of the solution.

The solvent was distilled off under reduced pressure, and then water wascompletely removed by azeotropic distillation with toluene. The residuewas recrystalized from methanol and ethyl acetate yieldingdibenzylphosphoryl-L-alanyl-L-prolyl-L-proline L-arginine salt crystals(620 mg) having a melting point 114° C. (s) to 122° C. (d) and specificrotatory power [α]_(D) ²⁰ =-73.3° (C=1.16, ethanol). A fixed weight ofthe sample of the product was heated in 6N hydrochloric acid at 110° C.for 17 hours, and from thus hydrolized material, amino acids analysiswas carried out. The ratio of alanine, proline and arginine was1.00:2.01:1.01.

Elementary analysis: Found C 50.65%, H 7.11%, N 12.30%; Calculation C50.17%, H 7.16%, N 12.41% as C₃₃ H₄₈ O₉ N₇ P.4H₂ O.

Median lethal dose (LD 50):

more than 15 g/kg in mouse

more than 15 g/kg in rat

Example 2

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-serine L-arginine salt:

Dibenzylphosphoryl-L-alanyl-L-proline (1.2 g, 2.5 m mole) as shown inthe Japanese patent first publication No. 104863/1981, HOBt (338 mg, 2.5m mole) and L-serine methylester hydrochloride (467 mg, 3 m mole) weresuspended in DMF (10 ml), and WSC (0.5 ml) was added dropwise theretowhile cooling to -15° C. and stirring. The reaction was carried out for3 hours under cooling and next overnight at a room temperature. Ethylacetate (100 ml) was added to the reaction solution, washed 1Nhydrochloric acid and water in order, and dried with anhydrous sodiumsulfate. The thus obtained solution was distilled under reduced pressureto give an oily dibenzylphosphoryl-L-alanyl-L-prolyl-L-serine methylester (1.3 g). The oily product gave a single spot with R_(f) 0.46 bythin layer chromatography (developing solvent;chloroform:methanol:acetic acid=95:5:3, colour forming method: sprayingof 25% hydrobromic acid and 0.1% ninhydrine and heating). The oilymaterial was dissolved in a mixture (20 ml) of acetone and methanol(1:1) and 1N sodium hydroxide (5 ml) was added thereto under coolingwith ice. The mixture was stirred for 1.5 hours at room temperature andneutralized with 1N hydrochloric acid. The solution was distilled underreduced pressure to eliminate the organic solvent, and the residue wasdissolved in ethyl acetate, washed with 1N hydrochloric acid and waterin order, and dried with anhydrous sodium sulfate. The thus obtainedsolution was distilled under reduced pressure to give oilydibenzylphosphoryl-L-alanyl-L-prolyl-L-serine (1.2 g). The oily materialgave a single spot with R_(f) 0.18 by thin layer chromatography(developing solvent: chloroform:methanol:acetic acid=95:5:3, colourforming method; spraying of 25% hydrobromic acid and 0.1% ninhydrine,and heating).

The oily material (380 mg) was dissolved in ethanol (5 ml) and theaqueous L-arginine solution (0.71 ml) as prepared above. The solutionwas distilled under the reduced pressure leaving a residue, and residualwater was removed completely by azeotropic distillation with toluene. Tothe residue, methanol was added and an insoluble material was separatedby filtration. The filtrate was distilled under reduced pressure to givean amorphous powder of dibenzylphosphoryl-L-alanyl-L-prolyl-L-serineL-arginine salt (230 mg).

A fixed weight of the sample was heated in 6N hydrochloric acid at 110°C. for 19 hours. From this hydrolized product, amino acids analysis wascarried out. The ratio of alanine, proline, serine and arginine was1.00:1.02:0.96:1.07.

Example 3

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-aspartic acid L-arginine salt:

Dibenzylphosphoryl-L-alanyl-L-proline (1.2 g, 2.5 m mole), HOBt (338 mg,2.5 m mole) and L-aspartic acid dibenzylester P-toluene sulfonic acidsalt (1.45 g, 3 m mole) were suspended in DMF (10 ml), and WSC (0.5 ml)was added dropwise thereto while cooling at -15° C. and stirring. Thereaction was carried out under cooling for 3 hours and thereafter atroom temperature overnight. To the reaction solution ethyl acetate (100ml) was added and thus obtained mixture was washed with 1N hydrochloricacid and water in order, and dried with anhydrous sodium sulfate. Thethus obtained solution was distilled under reduced pressure leaving aresidue to obtain oily dibenzylphosphoryl-L-alanyl-L-prolyl-L-asparaticacid dibenzylester (1.9 g). The oily material gave a single spot withR_(f) 0.75 by thin layer chromatography (developing solvent;chloroform:methanol:acetic acid=95:5:3, colour forming method: sprayingof 25% hydrobromic acid and 0.1% ninhydrine, and heating). This oilymaterial was dissolved in a mixture (20 ml) of acetone and methanol (1:1), and 1N sodium hydroxide (5 ml) was added thereto while cooling. Next,the mixture was stirred for 1.5 hours at room temperature andneutralized with 1N hydrochloric acid. The organic solvent was removedby distillation under reduced pressure, and the residue was dissolved inethyl acetate, washed with 1N hydrochloric acid and water and dried withanhydrous sodium sulfate. The solvent was removed by distillation underreduced pressure to obtain oilydibenzylphosphoryl-L-alanyl-L-prolyl-L-aspartic acid (1.6 g). The oilymaterial gave a single spot with R_(f) 0.15 by thin layer chromatography(developing solvent; chloroform:methanol:acetic acid=95:5:3, colourforming method: spraying of 25% hydrobromic acid and 0.1% ninhydrine,and heating).

The oily material (430 mg) was dissolved in ethanol (5 ml), and aqueousarginine solution (0.76 ml), which has been prepared by dissolvingL-arginine in water in the ratio of 1.74 g of L-arginine per 10 ml ofthe aqueous arginine solution, was added thereto. The solvent wasremoved by distillation under reduced pressure, and then the water wasremoved completely by azeotropic distillation with toluene. Methanol wasadded to the residue, and thus prepared insoluble material was separatedby filtration. The solvent was distilled off under reduced pressure toobtain amorphous powder ofdibenzylphosphoryl-L-alanyl-L-prolyl-L-aspartic acid L-arginine salt(390 mg).

The fixed weight of the sample was heated in 6N hydrochloric acid at110° C. for 19 hours. For thus obtained hydrolized material, amino acidsanalysis was carried out. The ratio of alanine, proline, aspartic acidand arginine was 1.00:0.97:1.04:1.04.

Example 4

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-tyrosine L-arginine salt:

Dibenzylphosphoryl-L-alanyl-L-proline (1.2 g, 2.5 m mole), HOBt (338 mg,2.5 m mole) and L-tyrosine ethylester hydrochloride (737 mg, 3 m mole)was suspended in DMF (10 ml), and WSC (0.5 ml) was added graduallythereto while cooling to -15° C. and stirring. The reaction was carriedout for 3 hours under cooling and then overnight at room temperature. Tothe reaction solution ethyl acetate (100 ml) was added, and the mixturewas washed with 1N hydrochloric acid and water in order, and dried withanhydrous sodium sulfate. The solvent was removed by distillation underreduced pressure to obtain an oilydibenzylphosphoryl-L-alanyl-L-prolyl-L-tyrosine ethylester (1.4 g). Thisoily material gave a single spot with R_(f) 0.50 by thin layerchromatography (developing solvent; chloroform:methanol:aceticacid=95:5:3, colour forming method: spraying of 25% hydrobromic acid and0.1% ninhydrine, and heating).

The oily material was dissolved in a mixture of acetone and methanol(1:1), and 1N sodium hydroxide (5 ml) was added thereto while cooling.Next, the mixture was stirred for 1.5 hours at room temperature, andneutralized with 1N hydrochloric acid. The thus obtained solution wasdistilled under reduced pressure leaving a residue and the residue wasdissolved in ethyl acetate, washed with 1N hydrochloric acid and waterand dried with anhydrous sodium sulfate. The thus dried solution wasdistilled under reduced pressure to obtain an oilydibenzylphosphoryl-L-alanyl-L-prolyl-L-tyrosine material (1.2 g). Theoily material gave a single spot with R_(f) 0.3 by thin layerchromatography (developing solvent; chloroform:methanol:aceticacid=95:5:3, colour forming method: spraying of 25% hydrobromic acid and0.1% ninhydrine, and heating).

The oily material (390 mg) was dissolved in ethanol (5 ml), and aqueousarginine solution (0.64 ml) prepared as above mentioned was addedthereto. The thus obtained solution was distilled under reduced pressureleaving a residue and water was removed completely by azeotropicdistillation with toluene. To the residue, methanol was added and thethus obtained insoluble material was removed by filtration, and thefiltrate was distilled leaving an amorphous powder ofdibenzylphosphoryl-L-alanyl-L-prolyl-L-tyrosine L-arginine salt (370mg). A fixed weight of the sample (the product) was heated in 6Nhydrochloric acid for 19 hours at 110° C., and amino acids in thushydrolized material were analyzed. The ratio of alanine, proline,tyrosine and arginine was 1.00:0.99:1.03:1.02.

Example 5

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-phenylalanyl L-arginine salt:

Dibenzylphosphoryl-L-alanyl-L-proline (1.2 g, 2.5 m mole), HOBt (338 mg,2.5 m mole) and L-phenylalanine ethylester hydrochloride (690 mg, 3 mmole) were suspended in DMF (10 ml) and WSC (0.5 ml) were added theretowhile cooling to -15° C. and stirring. The reaction was carried out for3 hours under cooling and then overnight at room temperature. To thereaction solution, ethyl acetate (100 ml) was added, and the mixture waswashed with 1N hydrochloric acid and water in order, and dried withanhydrous sodium sulfate. The the dried solution was distilled underreduced pressure to obtain an oilydibenzylphosphoryl-L-alanyl-L-prolyl-L-phenylalanine ethylester material(1.3 g). The oily material gave a single spot with R_(f) 0.7 by thinlayer chromatography (developing solvent; chloroform:methanol:aceticacid=95:5:3, colour forming method: spraying of 25% hydrobromic acid and0.1% ninhydrine, and heating).

The oily material was dissolved in a mixture (20 ml) of acetone andmethanol (1:1) and 1N sodium hydroxide (5 ml) was added under cooling.The mixture was stirred for 1.5 hours at room temperature, andneutralized with 1N hydrochloric acid. The thus neutralized solution wasdistilled under reduced pressure leaving a residue and the residue wasdissolved in ethyl acetate, washed with 1N hydrochloric acid and water,and dried with anhydrous sodium sulfate. The thus dried solution wasdistilled under reduced pressure leaving an oilydibenzylphosphoryl-L-alanyl-L-prolyl-L-phenylalanine (0.9 g). The oilymaterial gave a single spot with R_(f) 0.42 by thin layer chromatography(developing solvent; chloroform:methanol:acetic acid=95:5:3, colourforming method: spraying of 25% hydrobromic acid and 0.1% ninhydrine,and heating).

The oily material (280 mg) was dissolved in ethanol (5 ml), and theaqueous arginine solution (0.47 ml) as previously described was addedthereto. The hydrochloric solution was distilled under reduced pressureleaving a residue and water was completely removed by azeotropicdistillation with toluene. To the residue, methanol was added and thethus prepared insoluble matter was separated by filtration. The filtratewas distilled under reduced pressure leaving adibenzylphosphoryl-L-alanyl-L-prolyl-L-phenylalanine L-arginine saltmaterial (270 mg). The fixed weight of the sample was heated in 6Nhydrochloric acid for 19 hours at 110° C. In the thus hydrolizedmaterial amino acids were analyzed. The ratio of alanine, proline,phenylalanine and arginine was 1.00:1.00:1.08:1.05.

Example 6

Dibenzylphosphoryl-L-alanyl-L-prolyl-glycine L-arginine salt:

Dibenzylphosphoryl-L-alanyl-L-proline (1.2 g, 2.5 m mole), HOBt (338 mg,2.5 m mole) and glycine benzylester p-toluene sulfonic acid salt (1.0 g,3 m mole) were suspended in DMF (10 ml), and WSC (0.5 ml) was dropwiseadded thereto while cooling to -15° C. and stirring. The reaction wascarried out for 3 hours under cooling, and next, overnight at roomtemperature. To the reaction solution ethyl acetate (100 ml) was addedand the mixture was washed with 1N hydrochloric acid and water in order,and dried with anhydrous sodium sulfate. The thus dried solution wasdistilled under reduced pressure to give an oilydibenzylphosphoryl-L-alanyl-L-prolyl-glycine benzylester material (1.5g). The oily material gave a single spot with R_(f) 0.7 by thin layerchromatography (developing solvent; chloroform:methanol:aceticacid=95:5:3, colour forming method: spraying of 25% hydrobromic acid and0.1% ninhydrine and heating).

This oily material was dissolved in a mixture (20 ml) of acetone andmethanol (1:1) and 1N sodium hydroxide (5 ml) was added thereto undercooling. Next, the mixture was stirred for 1.5 hours at roomtemperature, and neutralized with 1N hydrochloric acid. The thusneutralized solution was distilled under reduced pressure leaving aresidue, and the residue was dissolved in ethyl acetate, washed with 1Nhydrochloric acid and water in order, and dried with anhydrous sodiumsulfate. The thus dried solution was distilled under reduced pressureleaving an oily dibenzylphosphoryl-L-alanyl-L-prolylglycine material(1.4 g). The oily material gave a single spot with R_(f) 0.4 by thinlayer chromatography (developing solvent; chloroform:methanol:aceticacid=95:5:3, colour forming method: spraying of 25% hydrobromic acid and0.1% ninhydrine and heating).

The oily material (610 mg) was dissolved in ethanol (5 ml) and theaqueous arginine solution as previously described (1.2 ml) was addedthereto. The hydrochloric solution was distilled under reduced pressure,and further water was removed completely by azeotropic distillation withtoluene leaving a residue. To the residue methanol was added and theinsoluble material was separated by filtration. The thus obtainedfiltrate was distilled under reduced pressure to obtain an amorphouspowder of dibenzylphosphoryl-L-alanyl-L-prolyl-glycine L-arginine saltmaterial (570 mg). The fixed weight of the sample was heated in 6Nhydrochloric acid for 19 hours at 110° C. In the hydrolized material,amino acids were analyzed. The ratio of alanine, proline, glycine andarginine was 1.00:0.91:1.00:1.11.

Example 7

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-valine L-arginine salt:

By using L-valine benzylester hydrochloride (15.3 m mole) in place ofL-proline benzylester hydrochloride (3.7 g, 15.3 m mole) as used inExample 1, the same experiment as in Example 1 is repeated and therebydibenzylphosphoryl-L-alanyl-L-prolyl-L-valine L-arginine salt can beproduced.

Example 8

Phosphoryl-L-alanyl-L-prolyl-L-proline L-arginine salt:

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-proline L-arginine salt (3 g, 4.2m mole), and L-arginine (1.45 g, 8.36 m mole) were dissolved in amixture of ethanol (50 ml) and water (10 ml), and palladium-carbon (1 g)was added thereto. After that catalytic reduction reaction was carriedout for 5 hours. The catalyst was removed by filtration and the solventwas removed by distillation. The residue was dissolved in water andfreeze-dried to obtain phosphoryl-L-alanyl-L-prolyl-L-prolinetri-L-arginine salt (3.63 g). After hydrolysis of the material with 6Nhydrochloric acid, amino acids were analyzed. The ratio of alanine,proline and arginine was 1.00:1.99:2.90.

Example 9

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-leucine L-arginine salt:

By using L-leucine benzylester hydrochloride (15.3 m mole) in place ofL-proline benzylester hydrochloride (3.7 g, 15.3 m mole) as used inExample 1, the same experiment as in Example 1 is repeated, and therebydibenzylphosphoryl-L-alanyl-L-prolyl-L-leucine L-arginine salt can beproduced.

Example 10

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-threonine,-L-methionine or-L-glutamic acid L-arginine salt:

By using hydroxyl group-protected-L-threonine, L-methionine orL-glutamic acid benzylester hydrochloride (15.3 m mole) in place ofL-proline benzylester hydrochloride (3.7 g, 15.3 m mole) as used inExample 1, the same experiments as in Example 1 are repeated and theprotective group is removed, if necessary, and thereby the titledobjective amino acid derivatives can be produced.

Example 11

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-proline amide:

Dibenzylphosphoryl-L-alanyl-L-proline (2.8 g, 6.4 m mole), HOBt (952 mg,7 m mole), and L-proline amide (805 mg, 7 m mole) were dissolved in DMF(10 ml), and N,N'-dimethylaminopropylethylcarbodiimide hydrochloride(1.34 g, 7 m mole) was added thereto while cooling to -15° C.

The reaction was continued for 3 hours under cooling and then overnightat room temperature, the DMF solution was distilled under reducedpressure, and the residue was dissolved in chloroform. The mixture waswashed with water, dried with anhydrous sodium sulfate, and the solventwas removed by distillation.

The residue was reprecipitated from methanol-ethyl ether to obtaindibenzylphosphoryl-L-alanyl-L-prolyl-L-prolineamide (890 mg). Meltingpoint: 89° C. The specific rotatory power [α]_(D) ²⁰ =-29.9° (C=0.4, 1Nacetic acid). After hydrolysis of the product with 6N hydrochloric acid,the amino acids were analyzed. The ratio of alanine, proline and ammoniawas 1.00:0.99:1.05.

Example 12

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-alanine L-arginine salt:

Dibenzylphosphoryl-L-alanyl-L-proline (1.4 g, 3.2 m mole),1-hydroxybenzotriazol (476 mg, 3.5 m mole) and L-alanine benzylesterp-toluene sulfonate (1.3 g, 3.5 m mole) were dissolved in DMF (5 ml),and WSC (0.64 ml) was added thereto while cooling to -15° C. Thereaction was continued for 3 hours under cooling and then overnight atroom temperature, and ethyl acetate (100 ml) was added thereto. Themixture was washed with 1N hydrochloric acid, 5% sodium bicarbonate andwater in order, and dried with anhydrous sodium sulfate. The thusobtained solution was distilled under reduced pressure leaving an oilydibenzylphosphoryl-L-alanyl-L-prolyl-L-alanine benzylester material.

Thus obtained oily material was dissolved in acetone (25 ml), and 1Nsodium hydroxide (4 ml) was added thereto while cooling with ice. Next,the mixture was stirred for 1 hour at room temperature and neutralizedwith 1N hydrochloric acid. The thus neutralized solution was distilledunder reduced pressure, and the residue was dissolved in ethyl acetate.The mixture was washed with 1N hydrochloric acid and water in order, anddried anhydrous sodium sulfate. The solution was distilled under reducedpressure, and the thus obtained oily material was dissolved in ethanol(3 ml). The aqueous arginine solution (1.2 ml) as described above, wasadded thereto, and the aqueous solution was distilled under reducedpressure. Further, water was removed completely by azeotropicdistillation with toluene. To the residue, ethanol was added andinsoluble material was separated by filtration. By adding ethyl acetateto the filtrate, an amorphous powder (500 mg) was obtained. Meltingpoint was 110°˜120° C.

Elementary analysis: Found C 52.11%, H 7.09%, N 13.69%; Calculation C52.19%, H 6.81%, N 13.74% as C₃₁ H₄₆ N ₁₁ O₉ P.1.2H₂ O.

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. The ratio of alanine, proline and arginine was1.94:1.03:1.00.

Example 13

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-arginine:

Dibenzylphosphoryl-L-alanyl-L-proline (1.8 g, 4 m mole), HOBt (540 mg),and L-arginine methylester 2 hydrochloric acid salt (2.62 g, 10 m mole)were dissolved in DMF (30 ml), and then triethylamine (2.8 ml, 20 mmole) and dicyclohexylcarbodiimide (1.03 g) were added thereto whilecooling to -15° C. The reaction was carried out for 4 hours undercooling and then overnight at room temperature. Thus produceddicyclohexylurea was separated by filtration. The solvent was distilledoff and 0.1N hydrochloric acid was added to the residue. The mixture waswashed with ethyl ether and the aqueous layer was absorbed with ionexchanging resin "Dia ion HP-20" as produced by MitsubishikaseikogyoCo., Inc. The resin was washed with water and the desired substanceswere eluted with 80% methanol (methanol:5% acetic acid=80:20). Theeluants were concentrated and the thus obtained residue was dissolved inacetone (10 ml). 0.1N sodium hydroxide (5 ml) was added to the mixtureunder cooling and the mixture was stirred for 1 hour at roomtemperature. The mixture was neutralized with 1N hydrochloric acid andthe thus neutralized solution was distilled under reduced pressureleaving a residue. The residue was dissolved in water and treated withion exchanging resins "Dia ion HP-20" for adsorption of the desiredsubstances. The resin was washed with water and the desired substanceswere eluted with 80% methanol, which is the same composition as above.The eluants were concentrated and the thus obtained residue wasdissolved in ethanol. To the solution ethyl ether was added to give anamorphous powder of dibenzylphosphoryl-L-alanyl-L-prolyl-L-arginine (800mg). The specific rotatory power was [α]_(D) ²⁰ =-36.5° (C=0.5,ethanol).

Elementary analysis: Found C 54.60%, H 6.81%, N 13.54%; Calculation C54.66%, H 6.62%, N 13.66% as C₂₈ H₃₉ N₆ O₇ P.0.7H₂ O.

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. Alanine:Proline:Arginine=1.00:1.01:1.00.

Example 14

Dibenzylphosphoryl-L-alanyl-L-prolyl-D-alanine L-arginine salt:

Dibenzylphosphoryl-L-alanyl-L-proline (1.4 g, 3.2 m mole), HOBt (476 mg,3.5 m mole) and D-alanine benzylester p-toluene sulfonic acid salt (1.3g, 3.5 m mole) were dissolved in DMF (5 ml), and WSC (0.64 ml) was addedthereto while cooling to -15° C. The reaction was carried out for 3hours under cooling and then overnight at room temperature. To thereaction solution ethyl acetate (100 ml) was added and the mixture waswashed with 1N hydrochloric acid, 5% sodium bicarbonate and water inorder. The mixture was dried with anhydrous sodium sulfate and the thusobtained solution was distilled under reduced pressure to give an oilydibenzylphosphoryl-L-alanyl-L-prolyl-D-alanine benzylester material.

The thus obtained oily material was dissolved in acetone (25 ml) and 1Nsodium hydroxide (4 ml) was added under cooling. Next, the mixture wasstirred for 1 hour at room temperature, and then neutralized with 1Nhydrochloric acid. The solvent was removed by distillation under reducedpressure and the residue was dissolved in ethyl acetate. The mixture waswashed with 1N hydrochloric acid and water, and dried with anhydroussodium sulfate. The solution was distilled and the residue was dissolvedin ethyl ether and n-hexan was added thereto to precipitate a crystal.The crystal was dissolved in ethanol and the aqueous arginine solutionas mentioned above (1.2 ml) was added. The aqueous solution wasdistilled under reduced pressure and further, water was completelyremoved by azeotropic distillation with toluene. To the residue, ethanolwas added, and a thus produced insoluble material was separated byfiltration. To the filtrate, ethyl ether was added to precipitatedibenzylphosphoryl-L-alanyl-L-prolyl-D-alanine L-arginine salt (1.3 g)crystals. Melting point: 82°˜85° C., Specific rotatory power [α]_(D) ²⁰=-31.1° (C=0.5, ethanol).

Elementary analysis: Found C 51.70%, H 6.93%, N 13.79%; Calculation C51.80%, H 6.87%, N 13.64% as C₃₁ H₄₆ N₇ O₉ P.1.5H₂ O.

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. Alanine:Proline:Arginine=2.00:1.03:0.97.

Example 15

Dibenzylphosphoryl-L-alanyl-L-prolyl-D-proline L-arginine salt:

Dibenzylphosphoryl-L-alanyl-L-proline (1.4 g, 3.2 m mole), HOBt (476 mg,3.5 m mole) and oily D-proline benzylester hydrochloride (968 mg, 4 mmole) were dissolved in DMF (5 ml) and WSC (0.64 ml, 3.5 m mole) wereadded thereto while cooling to -15° C. The reaction was carried out for3 hours under cooling and then overnight at room temperature, and ethylacetate (100 ml) was added to the reaction solution. The mixture waswashed with 1N hydrochloric acid, 5% sodium bicarbonate and water inorder, and then dried with anhydrous sodium sulfate. The solution wasdistilled under reduced pressure leaving an oilydibenzylphosphoryl-L-alanyl-L-prolyl-D-proline benzylester material.

The thus obtained oily material was dissolved in acetone (20 ml), and 1Nsodium hydroxide (4 ml) was added thereto under cooling. The mixture wasstirred for 2 hours at room temperature, and neutralized with 1Nhydrochloric acid. The thus neutralized solution was distilled underreduced pressure, and the residue was dissolved in ethyl acetate. Themixture was washed with 1N hydrochloric acid and water in order, andthen dried with anhydrous sodium sulfate. The solution was distilledunder reduced pressure, the residue was dissolved in ethanol (3 ml), andthe aqueous arginine solution as mentioned above (1.2 ml) was addedthereto. The thus obtained aqueous solution was distilled under reducedpressure, and further, water was completely removed by azeotropicdistillation with toluene. To the residue, ethanol was added andinsoluble material was separated by filtration. By adding ethyl ether tothe filtrate, an amorphous powder ofdibenzylphosphoryl-L-alanyl-L-prolyl-D-proline L-arginine salt (585 mg)was obtained.

Elementary Analysis: Found C 50.43%, H 7.09%, N 12.39%; Calculation C50.17%, H 7.16%, N 12.41% as C₃₃ H₄₈ N₇ O₉ P.4H₂ O.

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed.

Alanine:Proline:Arginine=1.00:2.08:1.01

Example 16

Dibenzylphosphoryl-L-alanyl-L-prolyl-trans-L-hydroxyproline L-argininesalt:

Dibenzylphosphoryl-L-alanyl-L-proline (1.4 g, 3.2 m mole), HOBt (476 mg,3.5 m mole), and trans-L-hydroxyproline benzylester p-toluene sulfonicacid salt (1.4 g, 3.5 m mole) were dissolved in DMF, and WSC (0.64 ml,3.5 m mole) was added thereto while cooling to -15° C. The reaction wascarried out for 3 hours under cooling and then overnight at roomtemperature. To the reaction solution ethyl acetate (100 ml) was addedand the mixture was washed with 1N hydrochloric acid, 5% sodiumbicarbonate and water in order, and dried with anhydrous sodium sulfate.The thus obtained solution was distilled under reduced pressure leavingan oily dibenzylphosphoryl-L-alanyl-L-prolyl-trans-L-hydroxyprolinebenzylester material.

Thus obtained oily material was dissolved in acetone (15 ml), and 1Nsodium hydroxide (4 ml) was added thereto under cooling with ice. Themixture was stirred for 2 hours at room temperature and neutralized with1N hydrochloric acid.

The solvent was distilled off under reduced pressure and the residue wasdissolved in water, and washed with ethyl acetate. The water solutionwas treated with ion exchanging resin "Dia ion HP-20" for adsorption ofdesired product. The resin was washed with water and the desired productwas eluated with 80% methanol (methanol:5% acetic acid=80:20). Theeluate was concentrated under reduced pressure and the residue wasdissolved in ethanol, and the aqueous arginine solution as mentionedabove (1.2 ml) was added thereto. The thus obtained aqueous solution wasdistilled under reduced pressure and further water was completelyremoved by azeotropic distillation with toluene. To the residue, ethanolwas added and an insoluble material was separated by filtration. Ethylether was added to the filtrate to obtain an amorphous powder ofdibenzylphosphoryl-L-alanyl-L-prolyl-trans-L-hydroxyproline L-argininesalt (450 mg). After hydrolysis of the product with 6N hydrochloricacid, amino acids were analyzed.Alanine:Proline:Hydroxyproline:Arginine=1.00:1.04:0.98:0.78.

Example 17

Dibenzylphosphoryl-L-alanyl-trans-L-hydroxyprolyl-L-proline:

A. t-butyloxycarbonyl-L-alanyl-trans-L-hydroxyproline

t-Butyloxycarbonyl-L-alanine (2.84 g, 1.5 m mole) andtrans-L-hydroxyproline benzylester p-toluene sulfonic acid salt (5.91 g,15 m mole) were dissolved in methylene dichloride (50 ml) and WSC (2.75ml, 15 m mole) was added thereto while cooling to -15° C. The reactionwas carried out for 2 hours under cooling and then for 3 hours at roomtemperature, and the methylene dichloride solution was distilled underreduced pressure leaving a residue. The residue was dissolved in ethylacetate (100 ml) and washed with 1N hydrochloric acid, 5% sodiumbicarbonate and water in order, and dried with anhydrous sodium sulfate.The solvent was distilled off under reduced pressure to obtain oilyt-butyloxycarbonyl-L-alanyl-trans-L-hydroxyproline benzylester.

The thus obtained oily material was dissolved in methanol (100 ml), andpalladium-carbon (500 mg) was added thereto. The reductive reaction wascarried out for 5 hours. The catalyst was removed by filtration and thesolvent was distilled off under reduced pressure. The residue wasrecrystalized with ethyl acetate-hexan to givet-butyloxycarbonyl-L-alanyl-trans-L-hydroxyproline (3.7 g).

B. t-butyloxycarbonyl-L-alanyl-trans-L-hydroxyprolyl-L-prolinebenzylester

t-Butyloxycarbonyl-L-alanyl-trans-L-hydroxyproline (3.02 g, 10 m mole)and L-proline benzylester hydrochloride (2.66 g, 11 m mole) weredissolved in methylene dichloride (50 ml) and WSC (2.01 ml, 11 m mole)was added thereto while cooling to -15° C. The reaction was carried outfor 3 hours at room temperature. The methylene dichloride solution wasdistilled under reduced pressure and the residue was dissolved in ethylacetate (100 ml), and washed with 1N hydrochloric acid, 5% sodiumbicarbonate and water in order, and dried with anhydrous sodium sulfate.The thus obtained solution was distilled under reduced pressure and theresidue was recrystalized with ethyl acetate-n-hexan to obtaint-butyloxycarbonyl-L-alanyl-trans-L-hydroxyprolyl-L-proline benzylester(4.1 g).

C. Dibenzylphosphoryl-L-alanyl-L-trans-L-hydroxyprolyl-L-proline

t-Butyloxycarbonyl-L-alanyl-L-trans-L-hydroxyprolyl-L-prolinebenzylester (2.93 g, 6 m mole) and trifluoroacetic acid (10 ml) weremixed, and stirred for 40 minutes. Trifluoroacetic acid was distilledoff under reduced pressure, and 4.8N hydrochloric acid dioxane solution(1.88 ml, 9 m mole) was added to the residue. The mixture was stirredwell and ethyl ether was added thereto.

Thus produced precipitate was obtained on the filter paper and driedover sodium hydroxide in the desiccator. The precipitate was dissolvedin DMF (3 ml) and neutralized by adding triethylamine (1.4 ml) whilecooling to -5° C. Dibenzylphosphorylchloride carbon tetrachloridesolution (10 m mole) and triethylamine were added thereto at not morethan 5° C. and at pH 8 to 9. The mixture was stirred for three hours anddistilled under reduced pressure until a residue was obtained. Theresidue was dissolved in ethyl acetate (100 ml) and washed with 1Nhydrochloric acid, 5% sodium bicarbonate and water in order, and driedwith anhydrous sodium sulfate. The solvent was removed by distillationunder reduced pressure to obtaindibenzylphosphoryl-L-alanyl-trans-L-hydroxyprolyl-L-proline benzylester.

Thus obtained oily material was dissolved in methanol (20 ml) and 1Nsodium hydroxide (6 ml) was added thereto while cooling with ice, andstirred for 3 hours at room temperature. The mixture was neutralizedwith 1N hydrochloric acid, and the solvent was distilled off underreduced pressure. The residue was dissolved in water and treated withion exchanging resin "Dia ion HP-20" for adsorption of the desiredproduct. The resin was washed with water, and the desired product waseluted with 80% methanol. The eluate was concentrated and the residuewas recrystalized with ethyl acetate-n-hexan to obtaindibenzylphosphoryl-L-alanyl-trans-L-hydroxyprolyl-L-proline (650 mg).After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. Alanine:Proline:Hydroxyproline=1.00:0.99:0.99.

Example 18

Dibenzylphosphoryl-L-isoleucyl-L-prolyl-L-proline L-arginine salt:

The oily t-Butyloxycarbonyl-L-isoleucyl-L-prolyl-L-proline benzylester(10 g, 20 m mole) and trifluoroacetic acid (50 ml) were mixed togetherunder cooling, and stirred for 50 minutes. The trifluoroacetic acidsolution was distilled under reduced pressure, and the residue wasdissolved in DMF (50 ml). The mixture was neutralized with triethylamine(3.5 ml) under cooling to -5° C., and thereafterdibenzylphosphorylchloride carbontetrachloride solution (30 m mole) andtriethylamine were dropwise added thereto at not more than 5° C. and atthe pH range 8-9. The mixture was stirred for 3 hours at roomtemperature and then distilled under reduced pressure. The residue wasdissolved in ethyl acetate (300 ml) and washed with 1N hydrochloricacid, 5% sodium bicarbonate and water in this order, and dried withanhydrous sodium sulfate. The thus obtained solution was distilled underreduced pressure to obtain an oilydibenzylphosphoryl-L-isoleucyl-L-prolyl-L-proline benzylester.

All the amounts of the oily material was dissolved in acetone (100 ml)and 1N sodium hydroxide (25 ml) was added thereto under cooling with iceand the mixture was stirred for 2 hours at room temperature. The mixturewas neutralized with 1N hydrochloric acid, and distilled under reducedpressure to obtain a residue. The residue was dissolved in ethylacetate. The mixture was washed with 1N hydrochloric acid and water inthis order, dried with anhydrous sodium sulfate and distilled underreduced pressure until a residue was obtained. The residue was dissolvedin ethanol, and the solution prepared by dissolving L-arginine (1.3 g)in water (3 ml) was added, and the resulting insoluble material wasseparated by filtration. The filtrate was distilled under reducedpressure leaving a residue.

The residue was recrystalized with ethanol-ethyl acetate to obtaindibenzylphosphoryl-L-isoleucyl-L-prolyl-L-proline L-arginine salt (4.3g). Melting point: 141° to 148° C. A specific rotatory power: [α]_(D) ²⁰=-84.9° (C=0.5, 50% ethanol).

Elementary analysis: Found C 53.28%, H 7.20%, N 12.65%; Calculation C53.11%, H 7.44%, N 12.05% as C₃₆ H₅₄ O₉ N₇ P.3H₂ O.

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. Isoleucine:Proline:Arginine=1.03:2.00:0.99

Example 19

Dibenzylphosphoryl-glycyl-L-prolyl-L-proline L-arginine salt:

Trifluoroacetic acid (10 ml) was added tot-butyloxycarbonylglycyl-L-prolyl-L-proline (1.85 g, 5 m mole) whilecooling and the mixture was stirred for 40 minutes. The trifluoroaceticacid solution was distilled under reduced pressure, and to the residue,ethyl ether was added. The thus produced precipitate was obtained on thefilter paper, and dissolved in DMF (10 ml). The mixture was neutralizedby adding triethylamine (1.5 ml) to the mixture while cooling to -5° C.,and dibenzylphosphorylchloride carbon tetrachloride solution (7 m mole)and triethylamine were dropwise added thereto while keeping atemperature not more than 5° C., and keeping a pH range to 8-9. Themixture was stirred for 3 hours at room temperature and distilled untila residue was obtained. The thus obtained residue was dissolved in ethylacetate (100 ml), washed with 1N hydrochloric acid and water in thisorder, and dried with anhydrous sodium sulfate. The solution wasdistilled and the residue was dissolved in ethanol, and L-arginine (690mg) water (2 ml) solution was added thereto. Water was removed bydistillation of the solvent and by azeotropic distillation with toluene.To the residue, ethanol was added and an insoluble material wasseparated by filtration. Ethyl ether was added to the filtrate to obtaindibenzylphosphorylglycyl-L-prolyl-L-proline L-arginine salt (430 mg).

After the hydrolysis of the product, amino acids were analyzed,Glycine:Proline:Arginine=1.00:1.96:0.96.

Example 20

Dibenzylphosphoryl-L-phenylalanyl-L-Prolyl-L-proline:

t-Butyloxycarbonyl-L-phenylalanyl-L-prolyl-L-proline benzylester (1.65g, 3 m mole) and trifluoroacetic acid (10 ml) were mixed together for 40minutes under cooling. The trifluoroacetic acid solution was distilledunder reduced pressure, and the residue was dissolved in DMF (5 ml). Themixture was neutralized by adding triethylamine (0.8 ml) thereto whilecooling to -5° C., and further dibenzylphosphorylchloride carbontetrachloride solution (5 m mole) were added dropwise while keeping atemperature to not more than 5° C. The pH range of reaction solution wasusually kept from 8 to 9 by adding triethylamine. The mixture wasstirred for 3 hours at room temperature, and then distilled until aresidue was obtained. The residue was dissolved in ethyl acetate (100ml) and washed with 1N hydrochloric acid, 5% sodium bicarbonate andwater in order, and dried with anhydrous sodium sulfate. The thusobtained solution was distilled under reduced pressure to obtain an oilydibenzylphosphoryl-L-phenylalanyl-L-prolyl-L-proline benzylestermaterial. The oily material was dissolved in acetone (20 ml) and 1Nsodium hydroxide (4 ml) was added thereto while cooling with ice and themixture was stirred for 3 hours at room temperature. Afterneutralization of the mixture with 1N hydrochloric acid, the thusneutralized solution was distilled under reduced pressure. The residuewas dissolved in ethyl acetate. The mixture was dried with 1Nhydrochloric acid and water in order, and dried with anhydrous sodiumsulfate. The thus obtained solution was distilled under reducedpressure, and the residue was reprecipitated with ethyl acetate andn-hexan to obtain dibenzylphosphoryl-L-phenylalanyl-L-prolyl-L-proline(820 mg).

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. Phenylalanine:Proline=0.99:2.00.

Example 21

Dibenzylphosphoryl-L-arginyl-L-prolyl-L-proline

Anisole (0.5 ml) was added toamyloxycarbonyl-tosyl-L-arginyl-L-prolyl-L-proline benzylester (1.45 g,2 m mole) and anhydrous hydrogen fluoride (20 ml) was added theretowhile cooling to -40° C. The reaction was carried out for 60 minutes at0° C., and anhydrous hydrogen fluoride solution was distilled underreduced pressure. The residue was washed with ethyl ether, and dissolvedin 1N acetic acid. The mixture was passed through adsorbent "Dowex 1×2"(type of CH₃ COO⁻) as produced by Dow Chemical Co. and the desiredproduct was eluated with water. The eluted was freeze-dried. Thusobtained amorphous L-arginyl-L-prolyl-L-proline was dissolved in DMF (8ml) and water (2 ml), and triethylamine (O.3 ml) was added thereto whilecooling to -5° C. for neutralization.

To the mixture dibenzylphosphorylchloride carbon tetrachloride solution(3 m mole) and triethylamine were added dropwise while keeping thetemperature to not more than 5° C., and keeping usually pH value to 8-9.The mixture was stirred for 3 hours at room temperature, then distilledoff under reduced pressure, and the residue was dissolved in water andthen passed through "Dowex 1×2" (a type of CH₃ COO⁻). The desiredproduct was eluted with water and the thus obtained eluate wasfreeze-dried. The thus obtained amorphous powder was further purified bycolumn chromatography with silica gel (eluating solution;chloroform:methanol:acetic acid=85:15:5). Main ingredients werecollected and freeze-dried to obtain amorphous powder ofdibenzylphosphoryl-L-arginyl-L-prolyl-L-proline (270 mg).

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. Arginine:Proline=1.04:2.00.

Example 22

Dibenzylphosphoryl-L-glutamyl-L-prolyl-L-proline

Anisole (0.5 ml) was added tot-butyloxycarbonyl-γ-benzyl-L-glutamyl-L-prolyl-L-proline (1.06 g, 2 mmole) and anhydrous hydrogen fluoride (20 ml) was added thereto whilecooling -40° C. The reaction was carried out for 1 hour at 0° C., andthen the anhydrous fluoride solution was distilled under reducedpressure. The residue was washed with ethyl ether and then dissolved in1N acetic acid, and the mixture was passed through ion exchange resin"IR-45" (CH₃ COO⁻). The desired product was eluated with water and theeluted was freeze-dried.

The thus obtained amorphous L-glutamyl-L-propyl-L-proline was dissolvedin DMF (8 ml) and water (2 ml), and neutralized by addition oftriethylamine (0.2 ml) while cooling to -5° C.Dibenzylphosphorylchloride carbon tetrachloride solution (3 m mole) andtriethylamine were added dropwise to the mixture while keeping thetemperature to not more than 5° C. and usually keeping the pH value to8-9. The mixture was stirred for 3 hours at room temperature, and thesolution was distilled under reduced pressure leaving a residue. Theresidue was dissolved in 5% sodium bicarbonate and then washed withethyl acetate. The aqueous phase was acidified with 1N hydrochloricacid, and the desired product was extracted with ethyl acetate. Theethyl acetate phase was washed with sodium chloride solution and driedwith anhydrous sodium sulfate. The ethyl acetate was distilled off underreduced pressure. The residue was re-precipitated with ethylacetate-ethyl ether to obtaindibenzylphosphoryl-L-glutamyl-L-prolyl-L-proline (140 mg).

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. Glutamic acid:Proline=0.92:2.00.

Example 23

Diethylphosphoryl-L-lysyl-L-prolyl-L-proline L-arginine salt:

A. t-Butyloxycarbonyl-ε-carbobenzoxy-L-lysyl-L-proline benzylester

t-Butyloxycarbonyl-ε-carbobenzoxy-L-lysine dicylohexylamine salt (10 g,17.8 m mole) was suspended in ethyl acetate, and washed with 1N sulfuricacid. Ethyl acetate phase was washed with water and dried with anhydroussodium sulfate, and ethyl acetate was distilled off under reducedpressure.

All the obtained oily t-butyloxycarbonyl-ε-carbobenzoxy-L-lysine andL-proline benzylester hydrochloride (4.3 g, 17.8 m mole) were dissolvedin methylene dichloride and WSC (3.3 ml, 17.8 m mole) was added theretowhile cooling to -15° C. The mixture was stirred overnight and themethylene dichloride solution was distilled under reduced pressureleaving a residue. The residue was dissolved in ethyl acetate. The ethylacetate solution was washed with 1N hydrochloric acid, 5% sodiumbicarbonate and water in order, and dried with anhydrous sodium sulfate.The ethyl acetate was distilled off under reduced pressure to obtain anoily t-butyloxycarbonyl-ε-carbobenzoxy-L-lysyl-L-proline benzylester.

All the thus obtained oily material was dissolved in methanol (20 ml)and 1N sodium hydroxide (20 ml) was added thereto under cooling. Themixture was stirred for 3 hours, and the aqueous solution was adjustedto pH 7 by addition of 1N hydrochloric acid. The methanol was distilledoff under reduced pressure. The aqueous solution was adjusted to pH 2 byaddition of 1N hydrochloric acid, and the desired product was extractedwith ethyl acetate. The ethyl acetate phase was washed with water anddried with anhydrous sodium sulfate. The ethyl acetate solution wasdistilled under reduced pressure leaving an oilyt-butyloxycarbonyl-ε-carbobenzoxy-L-lysyl-L-proline.

All the above oily material was dissolved in DMF (20 ml), and L-prolinebenzylester hydrochloride (4 g, 16.7 m mole), HOBt (2.2 g, 16.7 m mole)and WSC (3 ml, 16.7 m mole) were added thereto while cooling to -15° C.

The mixture was stirred overnight and excess of ethyl acetate was addedthereto. The ethyl acetate solution was washed with 1N hydrochloricacid, 5% sodium bicarbonate and water in order, and dried with anhydroussodium sulfate. The ethyl acetate solution was distilled under reducedpressure leaving an oilyt-butyloxycarbonyl-ε-carbobenzoxy-L-lysyl-L-prolyl-L-proline benzylesterresidue (11 g).

B. Diethylphosphoryl-L-lysyl-L-prolyl-L-proline L-arginine salt

Trifluoroacetic acid (50 ml) was added to the oilyt-butyloxycarbonyl-ε-carbobenzoxy-L-lysyl-L-prolyl-L-proline benzylester(11 g) under cooling and the mixture was stirred for 40 minutes.Trifluoroacetic acid was distilled off under reduced pressure and theresidue was dissolved in DMF (30 ml), and was neutralized by addition oftriethylamine, and then diethylphosphorylchloride carbon tetrachloridesolution (25 m mole) was added thereto while keeping the temperature tonot more than 5° C. and keeping usually pH value to 8-9. The mixture wasstirred for 4 hours at room temperature. The solvent was distilled offunder reduced pressure and the residue was dissolved in ethyl acetate,and washed with 1N hydrochloric acid, 5% sodium bicarbonate and water inorder. The solution was dried with anhydrous sodium sulfate, and thesolvent was distilled off under reduced pressure to obtain an oilydiethylphosphoryl-ε-carbobenzoxy-L-lysyl-L-prolyl-L-proline benzylester(9.5 g). The oily material (9.5 g) was dissolved in methanol (100 ml)and palladium-carbon (5 g) was added thereto, and thereafter catalyticreductive reaction was carried out for 4 hours. The catalyst was removedby filtration and the solution was distilled under reduced pressureleaving an oily diethylphosphoryl-L-lysyl-L-prolyl-L-proline residue.The oily material was dissolved in ethanol and L-arginine (2.3 g) water(4 ml) solution was added thereto. The solvent was distilled off underreduced pressure and water was removed by azeotropic distillation withtoluene. Ethanol was added to the residue, and the thus producedinsoluble material was removed by filtration. By adding ethyl acetate tothe filtrate, diethylphosphoryl-L-lysyl-L-prolyl-L-proline L-argininesalt (3.5 g) was obtained.

After hydrolysis of the product with 6N hydrochloric acid, the materialwas subjected to amino acid analysis.Lysine:Proline:Arginine=0.99:1.57:1.00.

Example 24

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-proline L-lysine salt:

L-lysine (701 mg, 4.8 m mole) was added to the oilydibenzylphosphoryl-L-alanyl-L-prolyl-L-proline (2.72 g, 5 m mole), andwater (40 ml) was added thereto to dissolve the solid substances, andthe solution was freeze-dried to obtain an amorphous powder ofdibenzylphosphoryl-L-alanyl-L-prolyl-L-proline L-lysine salt (3.4 g).

Elementary analysis: Found C 53.94%, H 7.50%, N 9.58%; Calculation C53.94%, H 7.27%, N 9.53% as C₃₃ H₄₈ N₅ O₉ P.2.5H₂ O.

Example 25

Diethylphosphoryl-L-alanyl-L-prolyl-L-proline L-arginine salt:

A. Diethylphosphorylchloride

Phosphorous acid diethylester (7 g, 50 m mole) was dissolved in carbontetrachloride solution (50 m mole, 70 ml), and to the solutionsulfurylchloride (6.8 g) carbon tetrachloride solution (25 ml) was addeddropwise under the temperature not more than 5° C. with passing ofnitrogen gas. After the addition, the mixture was stirred for 1.5 hourswith passing of nitrogen gas. The reaction was traced by thin layerchromatography (developing solvent, chloroform:ethanol:ethylacetate=5:2:5; Colour former, Iodine). It was confirmed that thestarting materials were did not remaind in the reaction solution, andthus produced diethylphosphorylchloride was used for next step reactionwithout further purification.

B. Diethylphosphoryl-L-alanyl-L-proline benzylester

Trifluoroacetic acid (50 ml) was added tot-butyloxycarbonyl-L-alanyl-L-proline benzylester (11.3 g, 30 m mole)under cooling and the mixture was stirred for 40 minutes.Trifluoroacetic acid was distilled off under reduced pressure and theresidue was dissolved in DMF and neutralized by addition oftriethylamine (8 ml) under cooling to -5° C. Next,diethylphosphorylchroride carbon tetrachloride solution (40 m mole) wasadded dropwisely thereto while keeping the temperature to not more than5° C., and keeping pH value to 8-9 and the mixture was stirred overnightat room temperature. The solution was distilled under reduced pressureleaving a residue, and the residue was dissolved in ethyl acetate. Thesolution was washed with 1N hydrochloric acid, 5% sodium bicarbonate andwater in order, and dried with anhydrous sodium sulfate. The solutionwas distilled under reduced pressure leaving an oilydiethylphosphoryl-L-alanyl-L-proline benzylester residue (9.2 g).

C. Diethylphosphoryl-L-alanyl-L-prolyl-L-proline L-arginine salt:

The oily diethylphosphoryl-L-alanyl-L-proline benzylester (8.5 g, 20.6 mmole) was dissolved in methanol and palladium-carbon (2 g) was addedthereto. The reductive reaction was carried out for 3 hours. Thecatalyst was removed by filtration and the solvent was distilled offunder reduced pressure to obtain an oilydiethylphosphoryl-L-alanyl-L-proline.

All the thus obtained oily material and L-proline benzylesterhydrochloride (5.3 g, 22 m mole) were dissolved in methylene dichloride(50 ml) and WSC (4 ml, 22 m mole) was added thereto while cooling to-15° C. The mixture was stirred for 3 hours under cooling and overnightat room temperature, and the solution was distilled under reducedpressure leaving a residue. The residue was dissolved in ethyl acetate,and washed with 1N hydrochloric acid, 5% sodium bicarbonate and water inthis order, and dried with anhydrous sodium sulfate. The solvent wasdistilled off under reduced pressure to obtain an oilydiethylphosphoryl-L-alanyl-L-prolyl-L-proline benzylester (9.7 g).

The thus obtained oily material was dissolved in methanol, andpalladium-carbon (2 g) was added thereto. The reductive reaction wascarried out for 3 hours. The catalyst was separated by filtration andthe solvent was distilled off under reduced pressure leaving a residue.The residue was purified by silica gel chromatography (solvent,chloroform:methanol:acetic acid=95:5:3) to obtain an oilydiethylphosphoryl-L-alanyl-L-prolyl-L-proline (3.1 g). The oily materialwas dissolved in ethanol and L-arginine (1.3 g) water (2 ml) solutionwas added thereto. The solution was distilled under reduced pressure toremove the solvent and water was removed by azeotropic distillation withtoluene. Ethanol was added to the residue and the thus obtainedinsoluble material was separated by filtration. By adding ethyl acetateto the filtrate, diethylphosphoryl-L-alanyl-L-prolyl-L-proline argininesalt (1.3 g) was obtained. Melting point: 98° to 102° C., Specificrotatory power [α]_(D) ²⁰ =-77.9° C. (C=0.65 ethanol).

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. Alanine:Proline:Arginine=1.00:2.04:0.80.

Example 26

N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-prolinebenzylester.

A. L-alanyl-L-prolyl-L-proline benzylester hydrochlorideN-t-butyloxycarbonyl-L-alanyl-L-prolyl-L-proline benzylester (6.0 g,12.7 m mole) as produced in the same manner as in Example 1, wasdissolved in 4.8N hydrogene chloride dioxane solution, and the mixturewas stirred for 1 hour at room temperature. The solvent was distilledoff under reduced pressure and ethyl ether was added to the residue toobtain L-alanyl-L-prolyl-L-proline benzylester hydrochloride incrystalline form (5.18 g, yield: 99.7%).

B. Dibenzylphenethylphosphonate

Dibenzyl hydrogene phosphite (12.3 g, 50 m mole) was dissolved in DMF(80 ml), and sodium hydride (60% in oil, 2.2 g) was added to the mixtureat an atmosphere of nitrogen while cooling to -15° C. and stirring. Thethus obtained mixture was stirred for 1.5 hours at the temperature notmore than 0° C. Phenethyl bromide (9.25 g, 50 m mole) in DMF (10 ml)solution was added dropwise to the above solution at an atmosphere ofnitrogen at the temperature not more than 0° C. The thus obtainedmixture was further reacted overnight at room temperature. DMF solutionwas distilled under reduced pressure leaving a residue and the residuewas dissolved in ethyl ether. The resulting solution was washed withwater, and dried with anhydrous sodium sulfate. The solution wasdistilled under reduced pressure leaving an oily dibenzylproduct. Theproduct was crystallized at room temperature and further recrystallizedwith ethyl ether to obtain dibenzylphenethylphosphonate (6.5 g, yield:35.5%) having melting point: 48° to 49° C.

B. Monobenzylphenethylphosphochloridate

Dibenzylphenethylphosphonate (3.19 g, 8.7 m mole) was dissolved incarbon tetrachloride (5 ml) and phosphorus pentachloride (1.91 g, 9.2 mmole) was added thereto under cooling in the ice bath, and the mixturewas stirred for 30 minutes. The mixture was heated to 70° C. in the rateof 10° C. per 15 minutes and stirred for 30 minutes at 70° C. Thereaction was carried out to completion for 2 hours at 70° C./1 mmHg inthe rotating evaporator, and then the solvent, phosphorus oxychlorideand benzyl chloride were distilled off. It was confirmed that thestarting materials were consumed thin layer chromatography on silica gel(developing solvent, benzene:ethyl acetate=1.1, Confirmation of Spot: UVlamp). The thus produced monobenzylphosphochloridate was used for thefollowing reaction without further purification.

C. N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-prolinebenzylester

L-alanyl-L-prolyl-L-proline benzylester hydrochloride (2.5 g, 6.1 mmole) was dissolved in methylene dichloride (20 ml) and triethyl amine(1.85 g, 18.3 m mole) was added thereto.Monobenzylphenethylphosphochloridate which had been produced fromdibenzylphenethylphosphonate (3.19 g, 8.7 m mole) was dissolved inmethylene dichloride (10 ml). This solution was added dropwise to theabove solution on an ice bath and the mixture was stirred overnight atroom temperature. The reaction solution was washed with water anddistilled under reduced pressure to eliminate the solvent. The residuewas dissolved in ethyl acetate and the mixture was washed with 1Nhydrochloric acid, water, 5% sodium bicarbonate and water in order, anddried with anhydrous sodium sulfate. The solution was distilled underreduced pressure leaving a sticky residue and it was purified by columnchromatography (silica gel, developing solvent; ethylacetate:methanol=20:1) to obtainN-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-proline benzylester (1.58g, yield: 41.7%). The product gave a single spot with R_(f) =0.4 by thinlayer chromatography (developing solvent; ethyl acetate:methanol=20:1,colour forming method: spraying of 25% hydrobromic acid and 0.1%ninhydrine and then heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.30 (2xd, 3H),1.70˜2.37 (m, 10H), 2.70˜3.10 (m, 2H), 3.30˜3.90 (m, 4H), 4.00˜4.35 (m,1H), 4.45˜4.75 (m, 2H), 4.90˜5.33 (m, 4H), 7.20 (S, 5H), 7.33 (S, 10H).

Example 27

N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-prolineL-arginine salt:

N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-prolinebenzylester (0.88 g, 1.4 m mole) was dissolved in acetone (5 ml) andsodium hydroxide (0.18 g, 4.5 m mole) in water (4 ml) solution was addedthereto under cooling in the ice bath, and the mixture was stirred for 2hours. To the solution water (100 ml) was added and the mixture waswashed with ethyl ether (50 ml). The solution was neutralized with 1Nhydrochloric acid, and the desired product was extracted with ethylether (100 ml). The ethyl ether solution was dried with anhydrous sodiumsulfate. The thus obtained solution was distilled under reduced pressureyielding an oilyN-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-proline material(0.54 g, yield: 71.6%). The thus obtained oily material (1.0 m mole) wasdissolved in ethanol (4 ml) and a solution of L-arginine (0.21 g, 1.0 mmole) in water (4 ml) was added thereto. The thus obtained solution wasdistilled under reduced pressure. By adding further water to it andfreeze-drying the mixture,N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-prolineL-arginine salt (0.59 g, yield: 60.2% fromN-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-prolinebenzylester) was obtained.

A fixed amount of the product (sample) was heated for 19 hours at 110°C. in 6N hydrochloric acid and the thus hydrolized material wassubjected to amino acid analysis. The ratio of alanine, proline andarginine obtained was 1.00:2.08:1.05.

Example 28

N-phenethylphosphonyl-L-alanyl-L-prolyl-L-proline 2 sodium salt:

N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-prolinebenzylester (0.67 g, 1.1 m mole) was dissolved in the mixture (24 ml) ofwater and methanol (1:1) and sodium bicarbonate (0.16 g, 2.2 m mole) wasadded thereto. Hydrogen gas was passed through the solution for 3 hoursunder atmospheric pressure at room temperature in the presence of 5%palladium carbon as a catalyst.

The catalyst was separated by filtration and the solution was distilledleaving a residue. By dissolving the residue in water and freeze-dryingit, N-phenethylphosphonyl-L-alanyl-L-prolyl-L-proline 2 sodium salt(0.54 g, quantitative yield) was obtained.

A fixed amount of the product was heated for 19 hours at 110° C. in 6Nhydrochloric acid. For the thus hydrolized material, amino acids wereanalyzed. The ratio of alanine and proline was 1.00:2.10.

NMR spectrum [D₂ O, internal standard: DSS] ppm: 1.25 (d, 3H), 1.50˜2.45(m, 10H), 2.60˜3.00 (m, 2H), 3.30˜3.90 (m, 4H), 4.00˜4.56 (m, 4H), 7.30(S, 5H).

Example 29

N-Phenethylphosphonyl-L-alanyl-N-(2-indanyl)glycyl-L-proline 2 sodiumsalt:

A. N-t-Butyloxycarbonyl-glycyl-L-proline benzylester

L-proline benzylester hydrochloride (20 g, 82.7 m mole) was added tochloroform (200 ml) and triethylamine (8.3 g, 82.7 m mole),N-t-butyloxycarbonyl-glycine (14.5 g, 82.7 m mole), and HOBt (11.2 g,82.7 m mole) were added thereto under cooling. To the mixture WSChydrochloride (15.82 g, 82.7 m mole) was added dropwise.

Further, the reaction was carried out for 2 hours on ice, and next,overnight at room temperature. The reaction solution was washed with 10%citric acid, aqueous sodium chloride, 5% sodium bicarbonate and aqueoussodium chloride in order, and dried with anhydrous sodium sulfate. Thesolution was distilled enough under reduced pressure leaving a stickyN-t-butyloxycarbonyl-glycyl-L-proline benzylester product (31.4 g, 83.5m mole; yield: 100%). The product gave a single spot with R_(f) =0.66 bysilica gel thin layer chromatography (developing solvent, benzene:ethylacetate=1.1, Colour forming method: spraying of 25% hydrobromic acid and0.1% ninhydrin and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.44 (S, 9H);1.83˜2.30 (m, 4H), 3.33˜3.72 (m, 2H), 3.90 (d, 2H), 4.44˜4.67 (m, 1H),5.12 (S, 2H), 5.42 (br, 1H), 7.32 (S, 5H).

B. Glycyl-L-proline benzylester hydrochloride

To N-t-butyloxycarbonyl-glycyl-L-proline benzylester (25.7 g, 68.2 mmole) 4N hydrogen chloride dioxane solution (170 ml) was added and thesolid material was dissolved. The mixture was stirred for 1 hour at roomtemperature. The mixture was evaporated under reduced pressure to obtaina half-solid glycyl-L-proline benzylester hydrochloride (20.2 g, 64.8 mmole, yield: 95%).

The product had a hygroscopicity and gave a single spot with R_(f) =0.27by silica gel thin layer chromatography (developing solvent,chloroform:methanol:triethylamine=40:2:1, colour forming method:spraying of 0.1% ninhydrin, and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.76˜2.26 (m, 4H),3.39˜3.80 (m, 2H), 3.98˜4.31 (m, 2H), 4.46˜4.73 (m, 1H), 5.06 (S, 2H),7.26 (S, 5H), 8.34 (br, 3H).

C. N-(2-indanyl) glycyl-L-proline benzylester

Glycyl-L-proline benzylester hydrochloride (20.0 g, 64.1 m mole) wasdissolved in dried ethanol (150 ml) and indan-2-on (4.23 g, 32.1 mmole), and Molecular Sieves (3A) (5 g) were added thereto. Sodiumcyanoborohydride (2.01 g, 32.1 m mole) was gradually added to the abovesolution while cooling with ice, and stirring. The mixture was stirredfor 1 hour under cooling with ice and then stirred for 2 hours at roomtemperature. A little insoluble material was separated by filtration.The ethanol was removed by distillation under reduced pressure from thereaction solution. To the residue water (150 ml) and ethyl ether (200ml) were added and the solution was adjusted to pH 1.5 with 6Nhydrochloric acid while stirring to dissolve it. The ethylether layerwas removed and the remaining water layer was washed with ethyl ether(200 ml). Next, to the water layer ethyl ether (400 ml) was added andthe solution was adjusted to pH 8.0 with sodium bicarbonate aqueoussolution. The desired product was extracted with ethyl ether and theethyl ether layer was separated. Further, the desired product wasextracted by the addition of ethyl ether (200 ml) to the water layer andthe thus extracted ethyl ether solution was separated. Such extractedethyl ether layers were combined together, and washed with the aqueoussolution saturated with sodium chloride (100 ml). The ethyl ether layerwas dried with anhydrous sodium sulfate, and concentrated under reducedpressure to obtain a crystal. The crystal was washed with n-hexane,obtained on the filter paper and dried to yield a white crystal ofN-(2-indanyl)glycyl-L-proline benzylester (8.02 g, 21.2 m mole, yield:33%). Melting point: 96°-98° C.

The product gave a single spot with R_(f) =0.77 and R_(f) =0.42,respectively by silica gel thin layer chromatography (Developingsolvent, n-butanol:acetic acid:water=4:2:2; andchloroform:methanol:triethylamine=40:2:1, Colour forming method:spraying of 0.1% ninhydrine, and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.82˜2.34 (m, 4H),2.70˜3.82 (m, 9H), 4.44˜4.72 (m, 1H), 5.14 (S, 2H), 7.13 (S, 4H), 7.32(S, 5H).

D. N-t-Butyloxycarbonyl-L-alanyl-N-(2-indanyl) glycyl-L-prolinebenzylester

N-(2-indanyl) glycyl-L-proline benzylester (6.0 g, 15.9 m mole),N-t-butyloxycarbonyl-L-alanine (3.0 g, 15.9 m mole) and HOBt (2.1 g,15.9 m mole) were dissolved in chloroform (60 ml) and the mixture wasstirred under cooling with ice, and WSC hydrochloride (3.03 g, 15.9 mmole) was added dropwise thereto. The reaction solution was left for 4days in a refrigerator. Next, to the reaction solution chloroform (100ml) was added and the mixture was washed with 10% citric acid, aqueoussodium chloride, 5% sodium bicarbonate and aqueous sodium chloride inthis order, and dried with anhydrous sodium sulfate. The solution wasdistilled under reduced pressure leaving a syrupy residue. The syrupyresidue was dissolved in ethyl ether (300 ml), and water (200 ml) wasadded thereto. The pH value was adjusted to 1.5 with 6N hydrochloricacid and the mixture was washed. The ethyl ether layer was separated andthen dried with anhydrous sodium sulfate.

Ethyl ether was removed by distillation under reduced pressure to obtainthe crystal. The crystalls were washed with n-hexane and recrystalizedwith ethyl acetate-isopropyl ether-n-hexan to obtain a white crystal ofN-t-butyloxycarbonyl-L-alanyl-N-(2-indanyl) glycyl-L-proline benzylester(3.02 g, 5.5 m mole, yield: 35%). Melting point: 135°-136° C.

The product gave a single spot with R_(f) =0.63 by silica gel thin layerchromatography (developing solvent; benzene:ethyl acetate=1:1, Colourforming method: spraying of 25% hydrobromic acid and 0.1% ninhydrin, andheating).

Unreacted starting materials of N-(2-indanyl) glycyl-L-prolinebenzylester (2.95 g) was recovered from the aqueous solution of pH 1.5produced by washing the above mentioned ethyl ether phase.

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.25 (d, 3H), 1.40 (S,9H), 1.65˜2.25 (m, 4H), 2.25˜3.85 (m, 7H), 3.90˜5.00 (m, 4H), 5.05 (S,2H), 5.40 (d, 1H), 7.10 (S, 4H), 7.23 (S, 5H).

E. L-Alanyl-N-(2-indanyl) glycyl-L-proline benzylester hydrochloride

N-t-butyloxycarbonyl-L-alanyl-N-(2-indanyl) glycyl-L-proline benzylester(2.72 g, 4.95 m mole) was dissolved in 4N hydrogen chloride dioxanesolution (50 ml) and the mixture was stirred for 1 hour at roomtemperature. The solvent was distilled under reduced pressure from thesolution, and thereby a sticky residue was obtained. After addition ofethyl ether the solution was filtered and a crystalline residue wasobtained on the filter paper, and immediately dried in a desiccator toobtain a white powder of L-alanyl-N-(2-indanyl) glycyl-L-prolinebenzylester hydrochloride (2.31 g, 4.76 m mole, yield: 96%). Thisproduct had a hygrospic nature, and gave a single spot with R_(f) =0.42by silica gel thin layer chromatography (developing solvent,chloroform:methanol:triethylamine=40:2:1, Colour forming method:spraying of 0.1% ninhydrin and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.20 (2xd, 3H),1.37˜2.45 (m, 4H), 2.50˜3.90 (m, 7H), 3.93˜5.00 (m, 4H), 5.10 (S, 2H),7.15 (S, 4H), 7.30 (S, 5H), 8.40 (br, 3H).

F. N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl)glycyl-L-proline benzylester

L-alanyl-N-(2-indanyl) glycyl-L-proline benzylester hydrochloride (1.5g, 3.09 m mole) was dissolved in methylene dichloride (15 ml), andtriethylamine (1.03 g, 10.2 m mole) was added thereto.Monobenzylphenethylphosphochloridate produced fromdibenzylphenethylphosphonate (1.61 g, 4.4 m mole) was dissolved inmethylene dichloride (10 ml). This solution was dropwise added to theabove solution under cooling with ice bath and further stirred for 2hours at room temperature. The reaction solution was washed with water,and the solvent was distilled off under reduced pressure. The residuewas dissolved in ethyl acetate, and the resulting solution was washedwith 1N hydrochloric acid, water, 5% sodium bicarbonate and water inthis order, and dried with anhydrous sodium sulfate. The solution wasdistilled under reduced pressure leaving a sticky residue. The residuewas purified by column chromatography (silica gel, developing solvent;ethyl acetate, methanol=20:1) to obtainN-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl)glycyl-L-proline benzylester (1.03 g, 1.46 m mole; yield: 47%).

This product gave a single spot with R_(f) =0.32, by thin layerchromatography (developing solvent, benzene:ethyl acetate=1:1, Colourforming method: spraying of 25% hydrobromic acid and 0.1% ninhydrin andheating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.20˜1.55 (m, 3H),1.65˜2.30 (m, 6H), 2.50˜4.70 (m, 13H), 4.80˜5.17 (m, 4H), 7.10 (S, 4H),7.15 (S, 5H), 7.25 (S, 5H), 7.30 (S, 5H).

G. N-Phenethylphosphonyl-L-alanyl-N-(2-indanyl) glycyl-L-proline 2sodium salt

N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl)glycyl-L-proline benzylester (0.74 g, 1.0 m mole) was dissolved in amixture (30 ml) of water and ethanol (2:3) and sodium bicarbonate (0.176g, 2.0 m mole) was added thereto. Hydrogen gas was passed through thesolution for 3 hours at room temperature and atmospheric pressure in thepresence of 5% palladium carbon as a catalyst. The catalyst wasseparated by filtration and the solution was distilled under reducedpressure until a residue was obtained. By dissolving the residue inwater and freeze-drying it, N-phenethylphosphonyl-L-alanyl-N-(2-indanyl)glycyl-L-proline 2 sodium salt (0.57 g, yield: 95.4%) was obtained.

NMR spectrum [D₂ O, internal standard: DSS] ppm: 1.05˜1.52 (m, 3H),1.55˜2.35 (m, 6H), 2.40˜ about 4.30 (m, 13H: peak masked partially withwater), 7.28 (S, 4H), 7.35 (S, 5H).

Example 30

N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl)glycyl-L-proline L-arginine salt:

A. N-t-Butyloxycarbonyl-glycyl-L-proline methylester L-prolinemethylester hydrochloride (13.6 g, 82.7 m mole) was suspended inchloroform (200 ml) and to the suspended solution triethylamine (8.3 g,82.7 m mole) was added while cooling. To the solution,N-t-butyloxycarbonyl glycine (14.5 g, 82.7 m mole) was added and WSChydrochloride (15.82 g, 82.7 m mole) was gradually added thereto whilecooling with ice. The reaction was carried out for 2 hours while coolingwith ice, and next overnight at room temperature. The reaction solutionwas washed with 10% citric acid, aqueous sodium chloride, 5% sodiumbicarbonate and aqueous sodium chloride in order, and dried withanhydrous sodium sulfate. The solution was distilled under reducedpressure leaving a syrupy N-t-butyloxycarbonyl-glycyl-L-prolinemethylester product (22.5 g, 78.5 m mole, yield: 95%). This product gavea single spot with R_(f) =0.48 by silica gel thin layer chromatography(developing solvent, benzene:ethyl acetate=1:1, colour forming method:spraying of 25% hydrobromic acid and 0.1% ninhydrin and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.44 (S, 9H),1.92˜2.32 (m, 4H), 3.45˜3.75 (m, 2H), 3.7 (S, 3H), 3.92 (d, 2H),4.40˜4.55 (m, 1H), 5.54 (br, 1H).

B. Glycyl-L-proline methylester hydrochloride

N-t-Butyloxycarbonyl-glycyl-L-proline methylester (21.3 g, 74.4 m mole)and 4N hydrogen chloride dioxan solution (150 ml) were mixed, and shakedto dissolve the solid material. It was stirred for 1 hour at roomtemperature. The solvent was well-distilled off under reduced pressureto obtain the solid glycyl-L-proline methylester hydrochloride (16.4 g,73.9 m mole, yield: 99%). The product was very hygroscopic and gave asingle spot with R_(f) =0.45 by silica gel thin layer chromatography(developing solvent, n-butanol:acetic acid:water=4:2:2, colour formingmethod: spraying of 0.1% ninhydrin and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.84˜2.40 (m, 4H),3.48˜4.27 (m, 4H), 3.67 (S, 3H), 4.34˜4.65 (m, 1H), 8.22 (br, 3H).

C. N-(2-indanyl) glycyl-L-proline methylester hydrochloride

Glycyl-L-proline methylester hydrochloride (16.0 g, 72.1 m mole) wasdissolved in methanol (150 ml), and Indan-2-on (4.75 g, 36.0 m mole) andtriethylamine (1.8 g, 18.0 m mole) were added thereto. Sodiumsyanoborohydride (2.27 g, 36.0 m mole) was gradually added thereto whilecooling with ice and stirring. The mixture was stirred for 1 hour whilecooling with ice and next for 5 hours at room temperature. A littleinsoluble material was separated by filtration, and the filtrate wasconcentrated under reduced pressure. To the residue, water (100 ml) andethyl ether (100 ml) were added, and the solution was adjusted to pH 1.5with 6N hydrochloric acid while stirring to dissolve it. The ethyl etherlayer was removed, and the remaining water layer was washed with ethylether (100 ml). The desired product was extracted from the water layerwith chloroform (100 ml×3 times). The thus obtained chloroform asextracted was concentrated under reduced pressure until a tar-likeresidue was obtained. To thus produced tar-like residue ethyl ether and5% sodium bicarbonate (200 ml) were added and the mixture was stirred todissolve the solid matter. The aqueous layer was removed and the ethylether layer was washed with an aqueous solution saturated with sodiumchloride. To the ethyl ether layer water (150 ml) was added, and thesolution was adjusted to pH 1.5 with 6N hydrochloric acid while stirringand ethyl ether was removed. The water layer was washed with ethyl ether(100 ml) and to the water layer chloroform (200 ml) was added. Thesolution was adjusted to pH 8.0 with sodium bicarbonate while stirring.The aqueous layer was removed, and chloroform layer was dried withanhydrous sodium sulfate, and the sodium sulfate was separated byfiltration. Separately, anhydrous hydrogen chloride gas was brown intothe chloroform, and thus obtained hydrogen chloride chloroform solutionwas added to the above mentioned chloroform layer. After checking toascertain that the pH of the solution had been preserved, a distillationwas carried out under reduced pressure, to yield N-(2indanyl)glycyl-L-proline methylester hydrochloride (4.50 g, 13.3 m mole, yield:37%).

This product gave a single spot respectively in the R_(f) =0.73 andR_(f) =0.58, by silica gel thin layer chromatography (developingsolvent, n-butanol:acetic acid:water=4:2:2, andchloroform:methanol:triethylamine=40:2:1, colour forming method:spraying of 0.1% ninhidrin and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.81˜2.38 (m, 4H),3.26 (d, 4H), 3.40˜3.93 (m, 4H), 3.66 (S, 3H), 4.03 (t, 1H), 4.40˜4.66(m, 1H), 5.29 (S, 2H), 7.16 (S, 4H)

D. N-t-Butyloxycarbonyl-L-alanyl-N-(2-indanyl) glycyl-L-prolinemethylester

N-(2-indanyl) glycyl-L-proline methylester hydrochloride (4.33 g, 12.8 mmole), N-t-butyloxycarbonyl-L-alanine (2.42 g, 12.8 m mole) and HOBt(1.73 g, 12.8 m mole) were dissolved in chloroform (50 ml), andtriethylamine (1.29 g, 12.8 m mole) was added thereto while cooling to-15° C. and then WSC hydrochloride (2.45 g, 12.8 m mole) was graduallyadded thereto under cooling with ice. Further, the reaction was carriedout for 2 hours on ice and next overnight at room temperature. Thereaction solution was washed with 10% citric acid, aqueous sodiumchloride, 5% sodium bicarbonate, and aqueous sodium chloride in order,and dried with anhydrous sodium sulfate. The solution was concentratedunder reduced pressure to obtain the syrupy material (3.88 g). It wasfurther purified by column chromatography (silica gel, developingsolvent, chloroform and next ethyl acetate) to obtainN-t-butyloxycarbonyl-L-alanyl-N-(2-indanyl) glycyl-L-proline methylester(2.42 g, yield: 64%). The product gave a single spot with R_(f) =0.4 bythin layer chromatography (developing solvent, ethylacetate:benzene=1:1, colour forming method: spraying of 25% hydrobromicacid and 0.1% ninhydrin and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.30 (d, 3H), 1.45 (S,9H), 1.75˜2.40 (m, 4H), 2.70˜3.85 (m, 7H), 3.70 (S, 3H), 3.90˜5.10 (m,4H), 5.45 (d, 1H), 72.0 (S, 4H).

N-(2-indanyl) glycyl-L-proline methylester hydrochloride (2.08 g) wasrecovered from the 10% citric acid solution which was used for washingthe above mentioned reaction mixture.

E. L-Alanyl-N-(2-indanyl) glycyl-L-proline methylester hydrochloride

N-t-Butyloxycarbonyl-L-alanyl-N-(2-indanyl) glycyl-L-proline methylester(2.24 g, 4.7 m mole) was dissolved in 4.8N hydrogen chloride dioxanesolution and stirred for 1 hour at room temperature. The solution wasdistilled off under reduced pressure leaving a residue and diethyl etherwas added to the residue to obtain L-alanyl-N-(2-indanyl)glycyl-L-prolyl methylester hydrochloride crystals (1.63 g, yield:84.1%).

NMR spectrum [D₂ O, internal standard: DSS] ppm:1.03˜1.37 (m, 3H), 1.60(br, 3H), 1.75˜2.55 (m, 4H), 2.55˜3.60 (m, 7H), 3.75 (S, 3H), 3.85˜about 4.5 (m, 4H, peak partially masked with water), 7.30 (S, 4H).

F. N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl)glycyl-L-proline methylester

L-Alanyl-N-(2-indanyl) glycyl-L-proline methylester hydrochloride (1.0g, 2.4 m mole) was dissolved in methylene dichloride (7 ml) andtriethylamine (0.77 g, 7.6 m mole) was added thereto.Monobenzylphenethylphosphochloridate produced fromdibenzylphenethylphosphate (1.33 g, 3.6 m mole) was dissolved inmethylene dichloride (3 ml). This solution was dropwise added to theabove solution while cooling in the ice bath. The mixture was stirredovernight at room temperature. To the reaction solution methylenedichloride (50 ml) was added, and thus obtained mixture was washed with1N hydrochloric acid, water, 5% sodium bicarbonate and water in orderand dried with anhydrous sodium sulfate. The solution was distilledunder reduced pressure leaving a sticky material (1.83 g). The thusobtained material was purified by column chromatography (silica gel,developing solvent, ethyl acetate, and next, ethylacetate:methanol=20:1) to obtainN-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-idanyl)glycyl-L-proline methylester (0.65 g, yield: 42.2%). The product gave asingle spot with R_(f) =0.25 by thin layer chromatography (developingsolvent, ethyl acetate, colour forming method: spraying of 25%hydrobromic acid and 0.1% ninhydrin and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.35 (m, 3H),1.70˜2.50 (m, 6H), 2.60˜3.85 (m, 9H), 3.65 (S, 3H), 3.85˜4.70 (m, 4H),4.97 (d, 2H), 5.47 (br, 1H), 7.20 (S, 9H), 7.30 (S, 5H).

G. N-(O-Benzyl-P-phenethylphosphonyl)L-alanyl-N-(2-indanyl)glycyl-L-proline

N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl)glycyl-L-proline methylester (0.58 g, 0.9 m mole) was dissolved inacetone (3 ml), and a sodium hydroxide solution (0.11 g, 2.8 m mole) inwater (3 ml) was added thereto, while cooling in the ice bath. Themixture was stirred for 1 hour. To this solution water (100 ml) wasadded and the mixture was washed with ethyl ether (50 ml). Theether-washed mixture was neutralized with 1N hydrochloric acid and thedesired product was extracted with ethyl acetate. The ethyl acetatelayer was dried with anhydrous sodium sulfate and the solvent wasdistilled off under reduced pressure to obtain a stickyN-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl)glycl-L-proline (0.53 g, yield: 93.5%).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.20˜1.50 (m, 3H),1.60˜2.32 (m, 4H), 2.40˜4.75 (m, 11H), 4.90 (d, 2H), 7.03 and 7.10 (2xS,9H), 7.23 (S, 5H).

H. N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl)glycyl-L-proline L-arginine salt

N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl)glycyl-L-proline (0.53 g, 0.9 m mole) was dissolved in ethanol (4 ml)and the aqueous solution produced by dissolving L-arginine (0.17 g, 0.9m mole) in water (4 ml) was added thereto. The solution was distilledunder reduced pressure leaving a residue and water was added thereto. Byfreeze-drying a,N-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-N-(2-indanyl)glycyl-L-proline L-arginine salt product (0.64 g, yield: 94.5%) wasobtained. The product gave a single spot with R_(f) =0.25 for peptide inthe free form and a single spot with R_(f) =0 for arginine,respectively, by thin layer chromatography (developing solvent,acetonitrile:acetic acid=30:1, Colour forming method: spraying of 25%hydrobromic acid and 0.1% ninhydrine, and heating).

Example 31

Dibenzylphosphoryl-L-alanyl-N-(2-indanyl)-glycyl-L-proline L-argininesalt:

A. Dibenzylphosphoryl-L-alanyl-N-(2-indanyl)-glycyl-L-prolinemethylester

L-alanyl-N-(2-indanyl) glycyl-L-proline methylester hydrochloride (1.44g, 3.5 m mole) was dissolved in chloroform (20 ml), and triethylamine(1.1 g, 10.9 m mole) was added thereto. Dibenzylphosphorylchloride,which had been produced from dibenzyl hydrogenphosphite (0.86 g, 3.5 mmole) in the same manner as described in Example 1, was dissolved incarbon tetrachloride (10 ml). This solution was dropwise added to theabove produced solution in the ice bath. The mixture was stirredovernight at room temperature. The thus precipitated triethyl aminehydrochloride was removed by filtration and the solvent was distilledoff under reduced pressure. The residue was dissolved in ethyl acetate,washed with 1N hydrochloric acid, water, 5% sodium bicarbonate and waterin order, and dried with anhydrous sodium sulfate. The solution wasdistilled under reduced pressure leaving a candy-like residue (1.64 g).This residue was purified by column chromatography (silica gel,developing solvent; ethyl acetate:methanol=80:1) to obtaindibenzylphosphoryl-L-alanyl-N-(2-indanyl)-glycyl-L-proline methylester(1.08 g, 48.5%). This product gave a single spot with R_(f) =0.66 bythin layer chromatography (Developing solvent, ethylacetate:methanol=80:1, Colour forming method: spraying of 25%hydrobromic acid and 0.1% ninhydrine, and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.10˜1.50 (m, 3H),1.65˜2.25 (m, 4H), 2.55˜3.82 (m, 7H), 3.65 (S, 3H), 3.85˜4.80 (m, 4H),5.00 (d, 4H), 5.45 (br, 1H), 7.16 (S, 4H), 7.35 (S, 10H).

C. Dibenzylphosphoryl-L-alanyl-N-(2-indanyl)-glycyl-L-prolineDibenzylphosphoryl-L-alanyl-N-(2-indanyl) glycyl-L-proline methylester(0.79 g, 1.2 m mole) was dissolved in acetone (5 ml), and sodiumhydroxide (0.15 g, 3.8 m mole) in water (4 ml) solution was addedthereto while cooling in the ice bath. The mixture was stirred for 1hour. To this solution water (100 ml) was added, and thereafter the thusformed solution was washed with ethyl ether (50 ml). The ether washedsolution was neutralized with 1N hydrochloric acid, and therefrom thedesired product was extracted with ethyl acetate. The thus extractedsolution was dried with anhydrous sodium sulfate, and the solvent wasdistilled off under reduced pressure to obtain an oilydibenzylphosphoryl-L-alanyl-N-(2-indanyl) glycyl-L-proline (0.77 g,yield: 99.7%).

NMR spectrum (CDCl₃, internal standard: TMS] ppm: 1.10˜1.55 (m, 3H),1.65˜2.30 (m, 4H), 2.40˜4.80 (m, 11H), 5.00 (d, 4H), 7.15 (S, 4H), 7.33(S, 10H).

D. Dibenzylphosphoryl-L-alanyl-N-(2-indanyl) glycyl-L-proline L-argininesalt

Dibenzylphosphoryl-L-alanyl-N-(2-indanyl)-glycyl-L-proline (0.77 g, 1.2m mole) was dissolved in ethanol (4 ml), and the aqueous solutionproduced by dissolving L-arginine (0.25 g, 1.2 m mole) in water (4 ml),was added thereto. The solvent was distilled off under reduced pressure,and then water was added thereto. This water solution was freeze-driedto obtain dibenzylphosphoryl-L-alanyl-N-(2-indanyl) glycyl-L-prolineL-arginine salt (0.84 g, yield: 85.2%). This product gave a single spothaving an R_(f) =0.2 for the peptide in free form, and an R_(f) =0 forarginine, respectively, by thin layer chromatography (developingsolvent, acetonitrile:acetic acid=30:1, Colour forming method: sprayingof 25% hydrobromic acid and 0.1% ninhydrine, and heating).

Example 32

Dibenzylphosphoryl-L-alanyl-L-prolyl-L-Proline potassium salt:

To the oily dibenzylphosphoryl-L-alanyl-L-prolyl-L-proline, (2.72 g, 5 mmole) 0.1N potassium hydroxide (50 ml) was added, and the thus producedsolution was freeze-dried to obtain an amorphous powder of thedibenzylphosphoryl-L-alanyl-L-prolyl-L-proline potassium salt (2.8 g).

Elementary analysis: Found C 51.58%, H 6.41%, N 6.79%; Calculation C51.60%, H 6.13%, N 6.69% as C₂₇ H₃₃ N₃ O₇ P.K.2.6H₂ O.

Example 33

Dibenzylphosphoryl-L-methionyl-L-prolyl-L-proline L-arginine salt:

A. Dibenzylphosphoryl-L-methionyl-L-prolyl-L-proline methylesterL-methionyl-L-prolyl-L-proline methylester hydrochloride (2.0 g, 5.1 mmole) was dissolved in methylene chloride (20 ml), and triethyl amine(1.54 g, 15.2 m mole) was added thereto. The solution was cooled at -30°C., and to the cooled solution, a carbon tetrachloride solution (10 ml)of dibenzylphosphorylchloride produced from dibenzylhydrogen phosphite(1.41 g, 5.1 m mole) was added dropwise, and the mixture was furtherstirred overnight at room temperature. To the reaction solution,methylene chloride (100 ml) was added, and the mixture was washed with1N hydrochloric acid, water, 5% sodium bicarbonate and water in thisorder. The mixture was dried with anhydrous sodium sulfate, and thesolvent was evaporated under reduced pressure to obtain a candy-likeresidue (3.23 g). The residue was purified by column chromatography(silica gel, developing solvent; ethyl acetate:methanol=20:1) to obtaindibenzylphosphoryl-L-methionyl-L-prolyl-L-proline methylester (1.87 g,yield: 59.6%). A single spot with R_(f) =0.1 was obtained by thin layerchromatography (developing solvent, ethyl acetate:methanol32 20:1,Colour forming method, spraying of 25% hydrobromic acid and 0.1ninhydrin, and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.55˜2.30 (m, 10H),2.03 (S, 3H), 2.60 (t, 2H), 3.30˜3.85 (m, 4H), 3.65 (S, 3H), 3.90˜4.25(br, 1H), 4.35˜4.65 (m, 3H), 5.00 (2xd, 4H), 7.30 (S, 10H).

B. Dibenzylphosphoryl-L-methionyl-L-prolyl-L-proline L-arginine salt

Dibenzylphosphoryl-L-methionyl-L-prolyl-L-proline methylester (1.87 g,3.0 m mole) was dissolved in acetone (18 ml), and 1N sodium hydroxide(4.5 ml) was added thereto under cooling and the mixture was stirred for4 hours. To the solution, water (100 ml) was added, and the solution waswashed with diethyl ether. Ethyl acetate (200 ml) was added thereto andthe mixture was adjusted to pH 2 with 1N hydrochloric acid whilestirring strongly. The ethyl acetate phase was separated and dried overanhydrous sodium sulfate. The solvent was evaporated to obtain an oilydibenzylphosphoryl-L-methionyl-L-prolyl-L-proline (1.81 g, yield:99.1%). All of the thus obtained product (3.0 m mole) was dissolved inethanol (15 ml) and a L-arginine (0.58 g, 3.0 m mole) water (5 ml)solution was added thereto. The solvent was evaporated, water was addedthereto, and the thus obtained solution was freeze-dried to obtaindibenzylphosphoryl-L-methionyl-L-prolyl-L-proline L-arginine salt (2.27g, yield: 98.7% from dibenzylphosphoryl-L-methionyl-L-prolyl-L-prolinemethylester). This product gave a single spot respectively having aR_(f) =0.8 for peptides in the free form and an R_(f) =0.25 for arginineby thin layer chromatography (developing solvent, n-butanol:aceticacid:water=2:1:1, Colour forming method, spraying of 25% hydrobromicacid and 0.1 ninhydrin, and heating). After hydrolysis of the productwith 6N hydrochloric acid, the amino acids were analyzed.Methionine:Proline:Arginine=1.00:2.03:0.95.

Example 34

N-Phenethylphosphonyl-L-methionyl-L-prolyl-L-proline 2 sodium salt:

A. N-(O-benzyl-P-phenethylphosphonyl)-L-methionyl-L-prolyl-L-prolinebenzylester

L-methionyl-L-prolyl-L-proline benzylester hydrochloride (5.69 g, 12.1 mmole) was dissolved in methylene chloride (30 ml), and triethylamine(3.85 g, 38.0 m mole) was added thereto. Methylene chloride solution (20ml) of monobenzylphenethyl phosphochloridate produced fromdibenzylphenethyl phosphonate (6.33 g, 17.3 m mole) was added dropwiseto the above solution while cooling in the coolant of -20° C. Further,it was stirred overnight at room temperature. To the reaction solution,methylene chloride (150 ml) was added, and the mixture was washed with1N hydrochloric acid, water, 5% sodium bicarbonate and water in order,and dried with anhydrous sodium sulfate. The solution was distilledunder reduced pressure to give a candy-like residue. This residue waspurified by column chromatography (Silica gel, developing solvent; ethylacetate:methanol=10:1) to giveN-(O-benzyl-P-phenethylphosphonyl)-L-methionyl-L-prolyl-L-prolinebenzylester (2.39 g, yield: 28.5%).

The product gave a single spot with R_(f) =0.7 by thin layerchromatography (developing solvent; ethyl acetate:methanol=10:1, Colourforming method: spraying of 25% hydrobromic acid and 0.1% ninhydrine,and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.60˜2.40 (m, 12H),2.10 (S, 3H), 2.45˜3.20 (m, 4H), 3.25˜3.90 (m, 4H), 3.95˜4.70 (m, 3H),4.80˜5.30 (m, 5H), 7.18 (S, 5H), 7.32 (S, 10H).

B. N-Phenethylphosphonyl-L-methionyl-L-prolyl-L-proline 2 sodium salt

N-(O-Benzyl-P-phenethylphosphonyl)-methionyl-L-prolyl-L-prolinebenzylester (1.195 g, 1.73 m mole) was dissolved in ethanol (15 ml), andsodium bicarbonate (0.290 g, 3.46 m mole) in water (5 ml) solution wasadded thereto. Hydrogen gas was passed through the above solution for 6hours at room temperature and atmospheric pressure in the presence of30% palladium barium carbonate as a catalyst. The catalyst was filteredoff and from the filtrate the solvent was evaporated. The thus obtainedresidue was dissolved in water and freeze-dried to obtainN-phenethylphosphonyl-L-methionyl-L-prolyl-L-proline 2 sodium salt (0.80g, yield: 83.3%). It gave a single spot with R_(f) =0.5 by thin layerchromatography (developing solvent; n-butanol:acetic acid:water=2:1:1,Colour forming method: spraying of 25% hydrobromic acid and 0.1%ninhydrine, and heating).

After hydrolysis of the product with 6N hydrochloric acid, the aminoacids were analyzed. Methionine:Proline=1.00:2.01.

NMR spectrum [D₂ O, internal standard: DSS] ppm: 1.50˜2.25 (m, 12H),2.12 (S, 3H), 2.70 (m, 4H), 3.40˜3.90 (m, 4H), 3.95˜4.40 (m, 3H), 7.30(S, 5H) FAB-MASS spectrum, m/e: 556 (M+1) 578 (M+Na).

Example 35

N-(O-Benzyl-P-phenethylphosphonyl)-L-methionyl-L-prolyl-L-prolineL-arginine salt:

N-(O-Benzyl-P-phenethylphosphonyl)-L-methionyl-L-prolyl-L-prolinebenzylester (1.195 g, 1.73 m mole) was dissolved in acetone (10 ml), and1N sodium hydroxide (2.6 ml) was added thereto under cooling in the icebath. The mixture was stirred for 5.5 hours. To the solution water (100ml) was added and the mixture was washed with diethyl ether, and thenethyl acetate (200 ml) was added thereto. The solution was adjusted topH 2 with 1N hydrochloric acid while stirring vigorously.

Ethyl acetate layer was separated and dried over anhydrous sodiumsulfate. The solvent was evaporated to give an oilyN-(O-benzyl-P-phenethylphosphonyl)-L-methionyl-L-prolyl-L-proline (1.02g, yield: 98.1%). All of thus obtained product (1.70 m mole) wasdissolved in ethanol (15 ml), and L-arginine (0.33 g, 1.70 m mole) water(5 ml) solution was added thereto. The solvent was distilled off underreduced pressure, and further, water was added thereto. The watersolution was freeze-dried to obtainN-(O-benzyl-P-phenethylphosphonyl)-L-methionyl-L-prolyl-L-prolineL-arginine salt yielding 94.0% based onN-(O-benzyl-P-phenethylphosphonyl)-L-methionyl-L-prolyl-L-prolinebenzylester. The product gave single spots respectively in R_(f) =0.7for peptides in the free form and R_(f) =0.2 for arginine, by thin layerchromatography (developing solvent; n-butanol:acetic acid:water=2:1:1,Color forming method; spraying of 25% hydrobromic acid and 0.1%ninhydrin, and heating).

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed.

Methionine:Proline:Arginine=1.00:2.05:0.89

Example 36

N.sup.α -Phenethylphosphonyl-L-lysyl-L-prolyl-L-proline 2 sodium salt:

A. N.sup.α -(O-Benzyl-P-phenethylphosphonyl)-N.sup.ε-benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylester

N.sup.ε -benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylesterhydrochloride (3.26 g, 5.4 m mole) was dissolved in methylene chloride(15 ml), and triethylamine (1.73 g, 17.1 m mole) was added thereto.Methylene chloride (10 ml) solution of monobenzylphenethylphosphochloridate produced from dibenzylphenethyl phosphonate (2.84 g,7.8 m mole) was added dropwise and the mixture was stirred overnight atroom temperature. To the reaction solution methylene chloride (100 ml)was added, and the mixture was washed with 1N hydrochloric acid, water,5% sodium bicarbonate and water in order, and dried with anhydroussodium sulfate. The solution was distilled under reduced pressureleaving a candy-like residue. The residue was purified with columnchromatography (Silica gel, developing solvent; ethylacetate:methanol=20:1) to obtain N.sup.α-(O-benzyl-P-phenethylphosphonyl)-N.sup.ε-benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylester (1.21 g,yield: 27.1%). The product gave a single spot with R_(f) =0.35 by thinlayer chromatography (developing solvent; ethyl acetate:methanol=20:1,Colour forming method: spraying of 25% hydrobromic acid and 0.1%ninhydrin, and heating).

B. N.sup.α -Phenethylphosphonyl-L-lysyl-L-prolyl-L-proline 2 sodium salt

N.sup.α -(O-Benzyl-P-phenethylphosphonyl)-N.sup.ε-benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylester (1.21 g, 1.5 mmole) was dissolved in methanol (18 ml), and sodium bicarbonate (0.247g, 2.9 m mole) water (5 ml) solution was added thereto. Hydrogen gas waspassed through the solution for 3 hours at room temperature andatmospheric pressure in the presence of 5% palladium carbon as acatalyst. The catalyst was removed by filtration. The solvent wasevaporated and the residue was dissolved in water. The solution wasfreeze-dried to obtain N.sup.α-phenethylphosphonyl-L-lysyl-L-prolyl-L-proline 2 sodium salt (0.84 gwhich is yielded quantitatively).

The product gave a single spot with R_(f) =0.3 by thin layerchromatography (developing solvent; n-butanol:acetic acid:water=2:1:1,Colour forming method: spraying of 25% hydrobromic acid and 0.1%ninhydrin, and heating).

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. Lysine:Proline=1.00:2.02

Example 37

N.sup.α -(O-Ethyl-P-phenethylphosphonyl)-L-lysyl-L-prolyl-L-prolineL-arginine salt:

A. Diethylphenethylphosphonate

Diethyl phosphite (27.64 g, 200 m mole) was dissolved in DMF (200 ml),and to the solution at nitrogen atmosphere while stirring and -15° C.,sodium hydride (60% in oil, 9.6 g, 220 m mole) was added. The mixturewas stirred for 1 hour at not more than 0° C. To the solution phenethylbromide (37.0 g, 200 m mole) was dropwise added and further the mixturewas stirred overnight at room temperature. The DMF solution wasdistilled under reduced pressure leaving a residue, and the residue wasdissolved in ethyl acetate. The ethyl acetate solution was washed withwater, and dried with anhydrous sodium sulfate. The solvent wasevaporated and the thus obtained oily product was distilled underreduced pressure to obtain diethylphenethylphosphonate (25.0 g, yield:51.6%) of boiling point 136.5°˜137.5° C./3 mmHg.

B. Monoethylphenethyl phosphochloridate

Diethylphenethyl phosphonate (1.73 g, 7.1 m mole) was dissolved incarbon tetrachloride (5 ml) and phosphorus pentachloride (1.55 g, 7.4 mmole) was added to the solution under cooling in the ice bath. Themixture was stirred for 30 minutes. The mixture was heated to 70° C. atthe rate of 10° C./15 minutes, and stirred for 30 minutes at 70° C. Thesolution was distilled under reduced pressure in the rotary evaporatorto eliminate the solvent and phosphorusoxychloride. Thus producedmonoethyl phenethyl phosphochloridate was used without furtherpurification for the following reaction.

C. N.sup.α -(O-Ethyl-P-phenethylphosphonyl)-N.sup.ε-benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylester

N.sup.ε -Benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylesterhydrochloride (3.0 g, 5.0 m mole) was dissolved in methylene chloride(15 ml), and triethylamine (1.59 g, 15.7 m mole) was added thereto.Methylene chloride (10 ml) solution of monoethylphenethylphosphochloridate produced from diethylphenethylphosphonate (1.73 g, 7.1m mole) was added dropwise to the above solution while cooling in thecoolant at -20° C. Further, the mixture was stirred overnight at roomtemperature. To the reaction solution methylene chloride (100 ml) wasadded, and the mixture was washed with 1N hydrochloric acid, water, 5%sodium bicarbonate and water in order and dried with anhydrous sodiumsulfate. The solution was distilled under reduced pressure leaving acandy-like residue. The residue was purified by column chromatography(Silica gel, developing solvent; ethyl acetate:methanol=20:1) to obtainN.sup.α -(O-ethyl-P-phenethyl phosphonyl)-N.sup.ε-benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylester (2.92 g,yield: 76.9%). The product gave a single spot with R_(f) =0.15 by thinlayer chromatography (developing solvent; ethyl acetate:methanol=20:1,Colour forming method: 25% hydrobromic acid and 0.1% ninhydrine, andheating).

D. N.sup.α -(O-Ethyl-P-phenethylphosphonyl)-L-lysyl-L-prolyl-L-prolineL-arginine salt

N.sup.α -(O-Ethyl-P-phenethylphosphonyl)-N.sup.ε-benzyloxycarbonyl-L-lysyl-L-prolyl-L-proline benzylester (1.46 g, 1.9 mmole) was dissolved in ethanol (10 ml), and L-arginine (0.40 g, 1.9 mmole) in water (5 ml) solution was added thereto. Hydrogen gas waspassed through the solution for 5 hours at atmospheric pressure and roomtemperature in the presence of 5% palladium carbon as a catalyst. Thecatalyst was removed by filtration and the solvent was evaporated. Thethus obtained residue was dissolved in water and freeze-dried to obtainN.sup.α -(O-ethyl-P-phenethylphosphonyl)-L-lysyl-L-prolyl-L-prolineL-arginine salt (1.06 g, yield: 77.7%). The product gave single spots,respectively having a R_(f) =0.55 for free peptides and R_(f) =0.2 forarginine by thin layer chromatography (developing solvent;n-butanol:acetic acid:water=2:1:2, Colour forming method: spraying of25% hydrobromic acid and 0.1% ninhydrine, and heating).

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed.

Lysine:Proline:Arginine=1.00:2.02:0.96

Example 38

N-n-Amylphosphonyl-L-alanyl-L-prolyl-L-proline 2 sodium salt:

A. Monobenzyl n-amylphosphochloridate

Dibenzyl n-amylphosphonate (2.74 g, 8.2 m mole) was dissolved in carbontetrachloride (5 ml), and phosphorus pentachloride (1.80 g, 8.6 m mole)was added to the solution while cooling in the ice bath, and the mixturewas stirred for 30 minutes. The mixture was heated to 70° C. at the rateof 10° C./15 minutes, and further stirred for 30 minutes at 70° C. Thesolution was distilled under reduced pressure in the rotary evaporatorto eliminate the solvent, phosphorus oxychloride and benzylchloride. Itwas confirmed that the starting materials did not remain in the reactionsolution by silica gel thin layer chromatography (developing solvent;benzene:ethyl acetate=1:1, Confirmation of spot: UV lamp). The thusproduced monobenzyl n-amylphosphochloridate was used without furtherpurification for the following reactions:

B. N-(O-benzyl-P-n-amylphosphonyl)-L-alanyl-L-prolyl-L-prolinebenzylester

L-Alanyl-L-prolyl-L-proline benzylester hydrochloride (2.37 g, 5.8 mmole) was dissolved in methylene chloride (15 ml), and triethylamine(1.84 g, 18.2 m mole) was added thereto. A methylene chloride (10 ml)solution of monobenzyl-n-amylphosphochloridate produced from dibenzyln-amylphosphonate (2.74 g, 8.2 m mole) was dropwise added to the abovesolution while cooling in the coolant at -15° C. The mixture was furtherstirred overnight at room temperature. To the reaction solution,methylene chloride (100 ml) was added, and the mixture was washed with1N hydrochloric acid, water, 5% sodium bicarbonate and water and driedover anhydrous sodium sulfate. The solvent was distilled off underreduced pressure to obtain a candy-like residue. It was purified bycolumn chromatography (Silica gel, developing solvent; ethylacetate:methanol=20:1) to obtainN-(O-benzyl-P-n-amylphosphonyl)-L-alanyl-L-prolyl-L-proline benzylester(1.26 g, yield: 36.5%). The product gave a single spot with R_(f) =0.2by thin layer chromatography (developing solvent; ethylacetate:methanol=20:1, Colour forming method: spraying of 25%hydrobromic acid and 0.1% ninhydrine, and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 0.87(t,3H),1.15˜1.50(m,7H), 1.55˜1.70(m,2H), 1.80˜2.40(m,10H), 3.23˜3.90(m,4H),3.95˜4.35(m,1H), 4.45˜4.70(m,2H), 4.85˜5.30(m,5H), 7.30(S,10H).

C. N-n-Amylphosphonyl-L-alanyl-L-prolyl-L-proline 2 sodium salt

N-(O-Benzyl-P-n-amylphosphonyl)-L-alanyl-L-prolyl-L-proline benzylester(1.26 g, 2.1 m mole) was dissolved in ethanol (20 ml), and sodiumbicarbonate (0.354 g, 4.2 m mole) in water (20 ml) solution was addedthereto. Hydrogen gas was passed through the solution at roomtemperature and atmospheric pressure for 3 hours in the presence of 5%palladium carbon as a catalyst. The catalyst was removed by filtration,and the solvent was evaporated. Thus obtained residue was dissolved inwater and freeze-dried to obtainN-n-amylphosphonyl-L-alanyl-L-prolyl-L-proline 2 sodium salt (0.93 g,yield: 95.6%). The product gave a single spot with R_(f) =0.3 by thinlayer chromatography (developing solvent; n-butanol:aceticacid:water=2:1:1, Colour forming method: spraying of 25% hydrobromicacid and 0.1% ninhydrine, and heating).

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. Alanine:Proline=1.00:1.99

NMR spectrum [D₂ O, internal standard: DSS] ppm: 0.86(t,3H),1.02˜1.70(m,9H), 1.75˜2.50(m,10H), 3.35˜4.35(m,7H).

Example 39

Diphenethylphosphinyl-L-alanyl-L-prolyl-L-proline sodium salt:

A. Diphenethyl phosphinic acid

Diethyl phosphite (6.9 g, 50 m mole) was added dropwise to the Grignardreagent solution produced from phenethylbromide (27.5 g, 148 m mole) andmagnesium (3.6 g, 148 m mole) in anhydrous diethyl ether (100 ml) whilecooling in the ice bath. The mixture was refluxed by heating overnight,and then the remaining Grignard reagent was decomposed by the additionof 1N hydrochloric acid (100 ml) and the diethyl ether was evaporated.Bromine was added to the solution while stirring in the ice bath to givea faintly brown coloured solution. The desired product was extractedwith chloroform from the solution. The chloroform layer was dried withanhydrous sodium sulfate, and the chloroform was distilled off underreduced pressure. The thus obtained residue was dissolved in hotethanol, and the solution was cooled to obtain diphenyl phosphinic acid(10.01 g, yield: 73.0%).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.80˜2.23 (m, 4H),2.70˜3.10 ppm (m, 4H), 7.18 (S, 10H), 11.70 (S, 1H).

B. Diphenethylphosphinylchloride

Diphenethylphosphinic acid (1.67 g, 6.1 m mole) was dissolved in carbontetrachloride (5 ml), and sulfuryl chloride (1.46 g, 10.8 ml) was addedthereto, and the mixture was heated while refluxing for 2 hours. It wasconfirmed that the starting material did not remain by tracement of theNMR spectrum. The solvent and excess sulfuryl chloride were distilledoff under reduced pressure to obtain an oilydiphenethylphosphinylchloride, which was supplied for the followingreaction.

C. Diphenethylphosphinyl-L-alanyl-L-prolyl-L-proline benzylester

L-Alanyl-L-prolyl-L-proline benzyl ester hydrochloride (2.0 g, 4.9 mmole) was dissolved in chloroform (20 ml), triethyl amine (1.36 g, 13.4m mole) was added thereto. Chloroform (8 ml) solution of diphenethylphosphinyl chloride produced from diphenethylphosphinic acid (1.67 g,6.1 m mole) was added dropwise to the above solution while cooling inthe coolant at -20° C. After the addition, the mixture was stirred for 1hour at not more than 0° C., and next, overnight at room temperature. Tothe reaction solution chloroform (100 ml) was added, and the mixture waswashed with 1N hydrochloric acid, water, 5% sodium bicarbonate and waterin order, and dried over anyhydrous sodium sulfate. The solvent wasdistilled off under reduced pressure to obtain the residue. The residuewas re-crystalized with ethyl acetate to obtain diphenethylphosphinyl-L-alanyl-L-prolyl-L-proline benzylester (1.19 g, yield:38.7%) of melting point 146°˜147° C. The product gave a single spot withR_(f) =0.3 by thin layer chromatography (developing solvent; ethylacetate:methanol=10:1, Colour forming method: spraying of 25%hydrobromic acid and 0.1% ninhydrine, and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.40 (d, 3H),1.75˜2.35 (m, 12H), 2.65˜3.05 (m, 4H), 3.20˜3.90 (m, 5H), 4.20 (q, 1H),4.50˜4.75 (m, 2H), 5.10 (q, 2H), 7.20 (S, 10H), 7.32 (S, 5H).

D. Diphenethylphosphinyl-L-alanyl-L-prolyl-L-proline sodium salt

N-Diphenethylphosphinyl-L-alanyl-L-prolyl-L-proline benzylester (0.77 g,1.2 m mole) was dissolved in methanol (20 ml), and sodium bicarbonate(0.103 g, 1.2 m mole) in water (10 ml) solution was added thereto.Hydrogen gas was passed through the above solution at room temperatureand atmospheric pressure for 3 hours in the presence of 5% palladiumcarbon as a catalyst. The catalyst was removed by filtration, and thesolvent was evaporated. The thus obtained residue was dissolved in waterand freeze-dried to obtain diphenethylphosphinyl-L-alanyl-L-prolyl-L-proline sodium salt (0.69 g, which isquantitative). The product gave a single spot with R_(f) =0.65 by thinlayer chromatography (developing solvent; n-butanol:aceticacid:water=2:1:1, Colour forming method; spraying of 25% hydrobromicacid and 0.1% ninhydrine, and heating).

After hydrolysis of the product with 6N hydrochloric acid, amino acidswere analyzed. Alanine:Proline=1.00:2.01.

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.40(d,3H),1.55˜2.35(m,12H), 2.60˜3.00(m,4H), 3.30˜3,80(m,4H), 3.90˜4.30(m,2H),4.40˜4.70(m,1H), 4.75˜5.20(br,1H), 7.20(S,10H).

Example 40˜52

    __________________________________________________________________________                         Example                                                                       No., to                                                                       which                                                    Ex-                  the same                          Amino Acids            am-                  reaction                          Analysis,              ple                  was  Starting         T.L.C.      Treatment of           No.                                                                              Product           applied                                                                            Materials        Rf value    6N                     __________________________________________________________________________                                                           HCl                    40 N--(O--ethyl-P--phenethylphos-                                                                  39   monoethylphenethylphosphochlo-                                                                 n-butanol:acetic                                                                          Met:Pro:Arg =             phonyl)-L-methionyl-L-prolyl-                                                                        ridate; L-methionyl-L-prolyl-                                                                  acid:water = 2:1:1                                                                        1.00:2.05:0.99            L-proline L-arginine salt                                                                            L-proline methylester hydrochlo-                                                               Rf = 0.65 (free peptide),                                    ride             Rf = 0.2 (L-Arginine)              41 N--(O--methyl-P--phenethylphos-                                                                 39   monomethylphenethylphosphochlo-                                                                n-butanol:acetic                                                                          Met:Pro:Arg =             phonyl)-L-methionyl-L-prolyl-                                                                        ridate; L-methionyl-L-prolyl-L-                                                                water = 2:1:1 Rf                                                                          1.00:2.01:0.97            L-proline L-arginine salt                                                                            proline methylester hydrochloride                                                              (free peptide), Rf = 0.2                                                      (L-arginine)                       42 N--(O--benzyl-P--phenethylphos-                                                                 26 and                                                                             monobenzylphenethylphosphochlo-                                                                n-butanol:acetic                                                                          Ala:Pro =                 phonyl)-L-alanyl-L-prolyl-L-                                                                    27   ridate; L-alanyl-L-prolyl-L-                                                                   water = 2:1:1 Rf                                                                          1.00:2.01                 proline sodium salt    proline methylester hydrochlor-                                               ride                                                43 N--(O--ethyl-P--phenethylphospho-                                                               39   monoethylphenethylphosphochlo-                                                                 n-butanol:acetic                                                                          Ala:Pro =                 nyl)-L-alanyl-L-prolyl-L-                                                                            ridate; L-alanyl-L-prolyl-L-                                                                   water = 2:1:1 Rf                                                                          1.00:2.04                 proline sodium salt    proline benzylester hydrochlo-                                                ride                                                44 Diethylphosphoryl-L-methio-                                                                     23   Diethylphosphorylchloride;                                                                     n-butanol:acetic                                                                          Met:Pro:Arg =             nyl-L-prolyl-L-proline L-methionyl-L-prolyl-L-proline                                                                 water = 2:1:1                                                                             1.00:2.02:0.98            L-argine salt          methylester hydrochloride                                                                      Rf = 7.0(free peptide)                                                        Rf = 0.2(L-arginine)               45 N--(O--ethyl-P--phenethylphos-                                                                  39   monoethylphenethylphosphochlo-                                                                 ethyl acetate:                                                                            Met:Pro =                 phonyl)-L-alanyl-L-prolyl-                                                                           ridate; L-alanyl-L-prolyl-L-                                                                   methanol = 20:1                                                                           1.00:2.05                 L-proline ethylester   proline ethylester hydrochlo-                                                                  Rf = 0.1                                                     ride                                                46 N--phenethylphosphonyl-L-                                                                       43   monobenzylphenethylphosphochlo-                                                                n-butanol:acetic:                                                                         Ala:Pro =                 alanyl-L-prolyl-L-proline                                                                            ridite; L-alanyl-L-prolyl-L-                                                                   water = 2:1:1                                                                             1.00:1.99                 ethylester sodium salt proline ethyl ester hydrochlo-                                                                 Rf = 0.5                                                     ride                                                47 N--phenethylphosphonyl-L-alanyl-                                                                26 and                                                                             monobenzylphenethylphosphochlo-                                                                n-butanol:acetic                                                                          Ala:Pro:Arg =             L-prolyl-L-proline                                                                              28   ridate; L-alanyl-L-prolyl-L-                                                                   water = 2:1:1                                                                             1.00:2.01:1.98            2 arginine salt        proline benzylester hydrochlo-                                                                 Rf = 0.4(free peptide)                                       ride             Rf = 0.2(L-arginine)               48 N--(3-phenylpropyl)phosphonyl-                                                                  26 and                                                                             monobenzyl(3-phenylprophyl)-                                                                   n-butanol:acetic                                                                          Ala:Pro =                 L-alanyl-L-prolyl-L-proline                                                                     28   phosphochoridate; L-alanyl-                                                                    water = 2:1:1                                                                             1.00:2.02                 2 sodium salt          L-prolyl-L-proline benzylester                                                                 Rf = 0.35                                                    hydrochloride                                       49 N--benzylphosphonyl-L-alanyl-                                                                   26 and                                                                             monobenzylbenzylphosphochlori-                                                                 n-butanol:acetic                                                                          Ala:Pro =                 L-prolyl-L-proline 2 sodium                                                                     28   date; L-alanyl-L-prolyl-L-pro-                                                                 water = 2:1:1                                                                             1.00:2.05                 salt                   line benzylester hydrochloride                                                                 Rf = 0.6                           50 N--(O--ethyl-P--phenethylphos-                                                                  39   monoethylphenethylphospho-                                                                     n-butanol:acetic                                                                          Lys:Pro =                 phonyl)-L-lysyl-L-prolyl-                                                                            chloridate; N.sup.ε --benzyloxycar-                                                    water = 2:1:1                                                                             1.00:2.05                 L-proline ethylester   bonyl-L-lysyl-L-prolyl-L-                                                                      Rf = 0.3                              hydrochloride          proline ethylester hydrochlo-                                                 ride                                                51 N--(O--ethyl-P--phenethyl-                                                                      39   monoethylphenethylphosphocholo-                                                                n-butanol:acetic                                                                          Lys:Pro =                 phosphonyl)-L-lysyl-L- ridate; N.sup.ε --benzyloxycarbonyl-                                                   water = 2:1:1                                                                             1.00:2.01                 prolyl-L-proline       L-lysyl-L-prolyl-L-proline                                                                     Rf = 0.55                                                    benzylester hydrochloride                           __________________________________________________________________________

Example 52

N-Phenethylphosphonyl-L-alanyl-L-prolyl-L-proline methylester sodiumsalt:

A. N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-prolinemethylester L-Alanyl-L-prolyl-L-proline methylester hydrochloride (3.3g, 10.0 mmole) was dissolved in methylene chloride (30 ml), andthiethylamine (3.03 g, 30.0 mmole) was added thereto.

Methylene chloride (20 ml) solution ofmonobenzylphenethylphosphochloridate produced fromdibenzylphenethylphosphonate (5.23 g, 14.3 mmole) was added dropwise tothe above solution while cooling in the ice bath. The mixture wasstirred overnight at room temperature. To the reaction solutionmethylene chloride (150 ml) was added, and the mixture was washed with1N hydrochloric acid, water, 5% sodium bicarbonate and water in order,and dried with anhydrous sodium sulfate. The solvent was distilled offunder reduced pressure to obtain a candy-like residue. It was purifiedby column chromatography (Silica gel, developing solvent; ethylacetate:methanol=10:1) to giveN-(O-benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-prolinemethylester (1.91 g, yield: 34.4%). The product gave a single spot withRf=0.2 by thin layer chromatography (developing solvent;ethylacetate:methanol=2:1, Colour forming method: spraying of 25%hydrobromic acid and 0.1% ninhydrine, and heating).

NMR spectrum [CDCl₃, internal standard: TMS] ppm: 1.33(2 x d, 3H),1.75˜2.40(m,10H), 2.60˜3.15(m,2H), 3.25˜3.90(m,4H), 3.68(S,3H),4.00˜4.35(br,1H), 4.50˜4.75(m,3H), 4.98(2 x d, 2H), 7.20(S,5H),7.35(S,5H).

B. N-Phenethylphosphonyl-L-alanyl-L-prolyl-L-proline methylester sodiumsalt

N-(O-Benzyl-P-phenethylphosphonyl)-L-alanyl-L-prolyl-L-prolinemethylester (1.21 g, 2.2 mmole) was dissolved in ethanol (20 ml), andsodium bicarbonate (0.18 g, 2.2 mmole) in water (5 ml) solution wasadded thereto. Hydrogen gas was passed through the above solution for 90minutes at room temperature and atmospheric pressure in the presence of5% palladium carbon as a catalyst. The catalyst was removed byfiltration, and the solvent was distilled off. The thus obtained residuewas dissolved in water, and the solution was freeze-dried to obtainN-phenethyl-phosphonyl-L-alanyl-L-prolyl-L-proline methylester sodiumsalt (0.95 g, yield: 89.5%). The product gave a single spot with Rf=0.45by thin layer chromatography (developing solvent; n-butanol:aceticacid:water=2:1:1, Colour forming method: spraying of 25% hydrobromicacid and 0.1% ninhydrine, and heating). NMR spectrum [CDCl₃, internalstandard: TMS] ppm: 1.20(d,3H), 1.50˜2.35(m,10H), 2.55˜3.05(m,2H),3.20˜4.10 (m,5H), 3.65(S,3H), 4.25˜4.90(m,3H), 7.10(s,5H).

Example 53

An antihypertensive activity of part of the amino acid derivativesprepared above was determined.

As subject animals there were used 2 SHR which had been trained enoughand been confirmed to be hypertensive spontaneously hypertenisive rats(male, 13 months of age, the body weight of 400˜440 g) per sample.

As an instrument for measuring the blood pressure there was usedProgrammed Electro-Sphygmomanometer PE-300 (Narco Co., U.S.A.), and theblood pressure was indirectly measured on conscious rats.

An aqueous solution or suspension of a sample (35 mg/kg) was onceforce-fed into the stomach by means of a peroral probe.

Table 1 shows the results.

                  TABLE 1                                                         ______________________________________                                        Systolic Blood Pressure, mmHg                                                                    4 hrs after                                                Sample  Pretreatment                                                                             administration                                                                             ΔBlood Pressure                         ______________________________________                                        Example                                                                       1       205        170          -35                                           2       202        180          -22                                           3       208        197          -11                                           4       207        195          -12                                           5       188        173          -15                                           6       212        190          -22                                           8       196        177          -19                                           12      193        182          -11                                           13      181        162          -19                                           14      183        161          -22                                           15      182        167          -15                                           16      181        162          -19                                           17      182        163          -19                                           18      192        171          -21                                           19      196        181          -15                                           20      184        169          -15                                           21      191        167          -24                                           22      206        196          -10                                           23      190        165          -25                                           24      192        161          -31                                           25      183        156          -27                                           26      182        160          -22                                           27      180        152          -28                                           28      183        151          -32                                           29      203        172          -31                                           30      184        154          -30                                           31      199        169          -30                                           32      185        160          -25                                           33      186        168          -18                                           34      183        154          -29                                           35      191        158          -33                                           36      200        177          -23                                           37      201        172          -29                                           38      187        167          -20                                           39      195        172          -23                                           40      196        166          -30                                           41      203        172          -31                                           42      194        180          -14                                           43      195        171          -24                                           44      189        179          -10                                           52      193        183          -10                                           *       198        193           -5                                           ______________________________________                                         *Dibenzyl phosphorylL-alanyl-L-proline Larginine salt                    

Example 54

Dry packed capsules containing 50 mg/capsule of an active ingredientwere prepared.

    ______________________________________                                        Dibenzylphosphoryl-L-alanyl-                                                                           50 mg                                                L-prolyl-L-proline L-arginate                                                 Lactose                  149 mg                                               Magnesium stearate       1 mg                                                 Capsule (size No. 1)     200 mg                                               ______________________________________                                    

An amino acid derivative was reduced to a No. 60 powder. Lactose andmagnesium stearate were passed through a No. 60 sieve cloth to fall overthe foregoing powder and mixed sufficiently with it. The mixture waspacked into No. 1 dry gelatin capsules.

Example 55

There were prepared tablets containingdibenzylphosphoryl-L-alanyl-L-prolyl-L-proline L-arginate (25 mg),pregelatinized starch (82 mg), microcrystalline cellulose (82 mg), andmagnesium stearate (1mg). In place of the foregoing amino acidderivative, other amino acid derivatives described hereinbefore could bemade into tablets together with pregelatinized starch, microcrystallinecellulose, and magnesium stearate.

Tablets for combined use containing a diuretic such ashydrochlorothiazide were prepared by incorporating thereindibenzylphosphoryl-L-alanyl-L-prolyl-L-proline-L-arginate (7.5 mg),hydrochlorothiazide (50 mg), pregelatinized starch (82 mg),microcrystalline cellulose (82 mg), and magnesium stearate (1 mg).

The foregoing results demonstrate that the amino acid derivatives of thepresent invention are useful as an antihypertensive drug or anintermediate for preparation thereof.

Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modificaitons can bemade thereto without departing from the spirit or scope of the inventionas set forth therein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. An amino acid derivative represented by theformula: ##STR8## wherein: R₁ and R₂ are the same or different from eachother, and each individually represents hydroxy, alkyloxy, aryloxy,aralkyloxy, alkyl, aryl, or aralkyl;R₃, R₅, R₈, and R₁₁, at least two ofwhich are the same or all of which are different from one another, andeach individually represents a hydrogen atom or lower alkyl; R₄, R₇, andR₁₀, at least two of which are the same, or all of which are differentfrom one another, and each individually represents a hydrogen atom, orlower alkyl, lower allyl, benzyl or indanyl, which may be furthersubstituted by HO, CO, carbamoyl, guanidino, imidazolyl, indolylmercapto or alkylthio; R₆ and R₉ are the same or different from eachother, and each individually represents hydrogen atom, lower alkyl,lower allyl, indanyl or benzyl; and; R₁₂ represents hydroxyl, alkyloxy,aryloxy, aralkyloxy, amino, mono- or dialkyl-, aryl- or aralkylamino;and R₆ and R₇ combined together, and R₉ and R₁₀ combined together, mayindependently form an alkylene bridge having 2 to 4 carbon atoms whichmay further contain a double bond or be further substituted withhydroxyl.
 2. The derivative as described in claim 1, wherein at leastone of amino acids constituting said amino acid derivative is in theL-form.
 3. The derivative as described in claim 1, wherein said aminoacid derivative is in the salt form.
 4. The derivative as described inclaim 1, wherein R₃, R₅, R₈ and R₁₁ are hydrogen atoms.
 5. Thederivative described in claim 1, wherein:R₁ and R₂ are the same ordifferent from each other, and each individually represents hydroxyl, alower alkyloxy having 1 to 5 carbon atoms, an aryloxy having 6 to 12carbon atoms, an aralkyloxy having 6 to 12 carbon atoms, a lower alkylhaving 1 to 5 carbons atoms, an aryl having 6 to 12 carbon atoms, or anaralkyl having 6 to 12 carbon atoms; R₃, R₅, R₈, and R₁₁, at least twoof which are the same, or all of which are different from one another,and each individually represents hydrogen atom or a lower alkyl having 1to 5 carbon atoms; R₄, R₇, and R₁₀, at least two of which are the same,or all of which are different from one another, and each individuallyrepresents a hydrogen atom, or lower alkyl having 1 to 5 carbon atoms,lower allyl having 1 to 5 carbon atoms, indanyl or benzyl, which may befurther substituted by HO, CO, carbomoyl, guanidino, imidazolyl,indolyl, mercapto or alkylthio, R₆ and R₉ are the same or different fromeach other, and each individually represents a hydrogen atom, a loweralkyl having 1 to 5 carbon atoms, an allyl having 6 to 12 carbon atoms,indanyl or benzyl; and R₁₂ represents hydroxyl, a lower alkyl having 1to 5 carbon atoms, an aryloxy having 6 to 12 carbon atoms, aralkyloxyhaving 6 to 12 carbon atoms, an amino, mono- or di-lower alkyl aminohaving 1 to 5 carbon atoms, aryl amino having 6 to 12 carbon atoms, oralkyl amino having 6 to 12 carbon atoms.
 6. The derivative described inclaim 1, wherein when an alkyl, an aryl or an aralkyl represented at R₄,R₇ and R₁₀ has at least one substituent, the substituent is an organicgroup selected from the group consisting of hydroxyl, carboxyl,carbamoyl, amino, guanidino, imidazolyl, indolyl, mercapto and loweralkylthio.
 7. The derivative described in claim 1, wherein R₆ and R₇combined together, or R₉ and R₁₀ claimed together, form an alkylenebridge having at least one substituent, the substituent is an organicgroup selected from the group consisting of hydroxyl, lower alkoxy,lower alkyl, oxo, condensed aryl ring and a condensed alicyclic ring. 8.The derivative described in claim 1, wherein:R₁ and R₂ are the same ordifferent from each other, and individually represent an organic groupselected from the group consisting of hydroxyl, methoxy, ethoxy,n-propyloxy, n-butyloxy, phenoxy, benzyloxy, phenethyloxy, methyl,ethyl, n-propyl, n-butyl, n-pentyl, phenyl, benzyl, phenethyl, andphenylpropyl; R₃, R₅, R₈ and R₁₁ represent a hydrogen atom, or at leastone of them represents methyl instead of the hydrogen atom; R₄, R₇ andR₁₀, at least two of which are the same, or all of which are differentfrom one another, each individually represents an organic group selectedfrom the group consisting of hydrogen atom, methyl, isobutyl, phenyl,benzyl, hydroxybenzyl, hydroxymethyl, carboxymethyl, carboxyethyl,carbamoylmethyl, carbamoylethyl, aminopropyl, aminobutyl, aminopentyl,guanidinopropyl, imidazolylmethyl, indolylmethyl, mercaptomethyl andmethylthioethyl; R₆ and R₉ are the same or different from each other,and each individually represents an organic group selected from thegroup consisting of hydrogen atom, methyl, cyclopropyl, cyclopentyl,cyclohexyl, 2-indanyl and 1-indanyl; and R₁₂ represents an organic groupselected from the group consisting of hydroxyl, methyloxy, ethyloxy,benzyloxy, amino, methylamino, ethylamino and phenyloxy.
 9. Thederivative as described in claim 1, wherein when R₆ and R₇ are combinedtogether, or R₉ and R₁₀ are combined together, the structure of thecombination is anyone of the following: ##STR9##
 10. The derivative asdescribed in claim 3, wherein said salt is one selected from the groupconsisting of sodium salt, potassium salt, lithium salt, ammonium salt,dicyclohexylamine salt, t-butylamine salt and basic amino acid salt. 11.An antihypertensive drug which comprises 0.2-2,000 mg of at least one ofthe amino acid derivatives represented by the formula: ##STR10## whereinR₁ and R₂ are the same or different from each other, and eachindividually represents hydroxyl, alkyloxy, aryloxy, aralkyloxy, alkyl,aryl, or aralkyl;R₃, R₅, R₈, and R₁₁, at least two of which are thesame, or all of which are different from one another, and eachindividually represents hydrogen atom or lower alkyl; R₄, R₇, and R₁₀,at least two of which are the same, or all of which are different fromone another, and each individually represents a hydrogen atom, or loweralkyl, lower allyl, benzyl or indanyl, which may be further substitutedby HO, CO carbamoyl, guanidino, imidazolyl, indolyl, mercapto oralkylthio; R₆ and R₉ are the same or different from each other, and eachindividually represents a hydrogen atom, lower alkyl, lower allyl,benzyl or indanyl; and R₁₂ represents hydroxyl, alkyloxy, aryloxy,aralkyloxy, amino, mono- or di-alkyl-, aryl-, or aralkylamino; and R₆and R₇ combined together, and R₉ and R₁₀ combined together, mayindependently form an alkylene bridge having 2 to 4 carbon atoms, whichmay further contain a double bond or be further substituted withhydroxyl and a pharmceutically acceptable carrier.
 12. Theantihypertensive drug described in claim 11, wherein at least one ofamino acids constituting said amino acid derivative is in the L-form.13. The antihypertensive drug described in claim 11, wherein said aminoacid derivative is in the form of a pharmaceutically acceptable salt.14. The antihypertensive drug described in claim 11, which furthercomprises a diuretic.